Methods and compositions for short stature plants through manipulation of gibberellin metabolism to increase harvestable yield

ABSTRACT

The present disclosure provides compositions and methods for altering gibberellin (GA) content in corn or other cereal plants. Methods and compositions are also provided for altering the expression of genes related to gibberellin biosynthesis through suppression, mutagenesis and/or editing of specific subtypes of GA20 or GA3 oxidase genes. Modified plant cells and plants having a suppression element or mutation reducing the expression or activity of a GA oxidase gene are further provided comprising reduced gibberellin levels and improved characteristics, such as reduced plant height and increased lodging resistance, but without off-types.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/847,283, filed Apr. 13, 2020, which is a divisional of U.S. patentapplication Ser. No. 15/679,699, filed Aug. 17, 2017 (now U.S. Pat. No.10,724,047), which claims the benefit of U.S. Provisional ApplicationNo. 62/376,298, filed Aug. 17, 2016, U.S. Provisional Application No.62/442,377, filed Jan. 4, 2017, and U.S. Provisional Application No.62/502,313, filed May 5, 2017, all of which are incorporated byreference herein in their entireties.

INCORPORATION OF SEQUENCE LISTING

A sequence listing contained in the file named “P34494US07_SL.txt” whichis 293,557 bytes (measured in MS-Windows®) and was created on Jun. 22,2022, is filed electronically herewith and incorporated by reference inits entirety.

BACKGROUND Field

The present disclosure relates to compositions and methods for improvingtraits, such as lodging resistance and increased yield, in monocot orcereal plants including corn.

Related Art

Gibberellins (gibberellic acids or GAs) are plant hormones that regulatea number of major plant growth and developmental processes. Manipulationof GA levels in semi-dwarf wheat, rice and sorghum plant varieties ledto increased yield and reduced lodging in these cereal crops during the20^(th) century, which was largely responsible for the Green Revolution.However, successful yield gains in other cereal crops, such as corn,have not been realized through manipulation of the GA pathway. Indeed,some mutations in the GA pathway genes have been associated with variousoff-types in corn that are incompatible with yield, which has ledresearchers away from finding semi-dwarf, high-yielding corn varietiesvia manipulation of the GA pathway.

There continues to be a need in the art for the development of monocotor cereal crop plants, such as corn, having increased yield and/orresistance to lodging.

SUMMARY

In a first aspect, the present disclosure provides a recombinant DNAconstruct comprising a transcribable DNA sequence encoding a non-codingRNA molecule, wherein the non-coding RNA molecule comprises a sequencethat is at least 80%, at least 85%, at least 90%, at least 95%, at least96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100%complementary to at least 15, at least 16, at least 17, at least 18, atleast 19, at least 20, at least 21, at least 22, at least 23, at least24, at least 25, at least 26, or at least 27 consecutive nucleotides ofa mRNA molecule encoding an endogenous GA oxidase protein in a monocotor cereal plant or plant cell, the endogenous GA oxidase protein beingat least 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, at least 99.5%, or 100% identicalto SEQ ID NO: 9, 12, 15, 30 or 33, and wherein the transcribable DNAsequence is operably linked to a plant-expressible promoter.

In a second aspect, the present disclosure provides a recombinant DNAconstruct comprising a transcribable DNA sequence encoding a non-codingRNA molecule, wherein the non-coding RNA molecule comprises a sequencethat is at least 90%, at least 95%, at least 96%, at least 97%, at least98%, at least 99%, at least 99.5%, or 100% complementary to at least 15,at least 16, at least 17, at least 18, at least 19, at least 20, atleast 21, at least 22, at least 23, at least 24, at least 25, at least26, or at least 27 consecutive nucleotides of a mRNA molecule encodingan endogenous GA20 oxidase protein in a monocot or cereal plant or plantcell, the endogenous GA20 oxidase protein being at least 80%, at least85%, at least 90%, at least 95%, at least 96%, at least 97%, at least98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 9,and wherein the transcribable DNA sequence is operably linked to aplant-expressible promoter.

In a third aspect, the present disclosure provides a recombinant DNAconstruct comprising a transcribable DNA sequence encoding a non-codingRNA molecule, wherein the non-coding RNA molecule comprises a sequencethat is at least 90%, at least 95%, at least 96%, at least 97%, at least98%, at least 99%, at least 99.5%, or 100% complementary to at least 15,at least 16, at least 17, at least 18, at least 19, at least 20, atleast 21, at least 22, at least 23, at least 24, at least 25, at least26, or at least 27 consecutive nucleotides of a mRNA molecule encodingan endogenous GA20 oxidase protein in a monocot or cereal plant or plantcell, the endogenous GA20 oxidase protein being at least 80%, at least85%, at least 90%, at least 95%, at least 96%, at least 97%, at least98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 15,and wherein the transcribable DNA sequence is operably linked to aplant-expressible promoter.

In a fourth aspect, the present disclosure provides a recombinant DNAconstruct comprising a transcribable DNA sequence encoding a non-codingRNA molecule, wherein the non-coding RNA molecule comprises a sequencethat is at least 90%, at least 95%, at least 96%, at least 97%, at least98%, at least 99%, at least 99.5%, or 100% complementary to at least 15,at least 16, at least 17, at least 18, at least 19, at least 20, atleast 21, at least 22, at least 23, at least 24, at least 25, at least26, or at least 27 consecutive nucleotides of a mRNA molecule encodingan endogenous GA3 oxidase protein in a monocot or cereal plant or plantcell, the endogenous GA3 oxidase protein being at least 80%, at least85%, at least 90%, at least 95%, at least 96%, at least 97%, at least98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 30 or33, and wherein the transcribable DNA sequence is operably linked to aplant-expressible promoter.

In a fifth aspect, the present disclosure provides a recombinant DNAconstruct comprising a transcribable DNA sequence encoding a non-codingRNA molecule, wherein the non-coding RNA molecule comprises a sequencethat is at least 90%, at least 95%, at least 96%, at least 97%, at least98%, at least 99%, at least 99.5%, or 100% complementary to at least 15,at least 16, at least 17, at least 18, at least 19, at least 20, atleast 21, at least 22, at least 23, at least 24, at least 25, at least26, or at least 27 consecutive nucleotides of a mRNA molecule encodingan endogenous GA20 oxidase protein in a monocot or cereal plant or plantcell, the endogenous GA20 oxidase protein being at least 80%, at least85%, at least 90%, at least 95%, at least 96%, at least 97%, at least98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 12,and wherein the transcribable DNA sequence is operably linked to aplant-expressible promoter.

In a sixth aspect, the present disclosure provides a recombinant DNAconstruct comprising a transcribable DNA sequence encoding a non-codingRNA molecule, wherein the non-coding RNA molecule comprises a sequencethat is at least 80%, at least 85%, at least 90%, at least 95%, at least96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100%complementary to at least 15, at least 16, at least 17, at least 18, atleast 19, at least 20, at least 21, at least 22, at least 23, at least24, at least 25, at least 26, or at least 27 consecutive nucleotides ofa mRNA molecule encoding an endogenous protein in a monocot or cerealplant or plant cell, the endogenous protein being at least 80%, at least85%, at least 90%, at least 95%, at least 96%, at least 97%, at least98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 86,90, 94, 97, 101, 104, 108, 112, 116, 118, 121, 125, 129, 133, or 136,and wherein the transcribable DNA sequence is operably linked to aplant-expressible promoter. In a further aspect, the present disclosurealso provides a transformation vector comprising a recombinant DNAconstruct disclosed herein. In a further aspect, the present disclosurealso provides a transgenic monocot or cereal plant, plant part or plantcell comprising a recombinant DNA construct disclosed here. In oneaspect, a transgenic corn plant, plant part or plant cell is provided.In another aspect, a method is provided for producing a transgeniccereal plant, comprising: (a) transforming at least one cell of anexplant with a recombinant DNA construct disclosed herein, and (b)regenerating or developing the transgenic cereal plant from thetransformed explant. In another aspect, a cereal plant is transformedvia Agrobacterium mediated transformation or particle bombardment.

In a seventh aspect, the present disclosure provides a method forlowering the level of at least one active GA molecule in the stem orstalk of a corn or cereal plant comprising: suppressing one or more GA3oxidase or GA20 oxidase genes with a recombinant DNA construct in one ormore tissues of the transgenic cereal or corn plant.

In an eighth aspect, the present disclosure provides a transgenic cornor cereal plant comprising a recombinant DNA construct, wherein therecombinant DNA construct comprises a transcribable DNA sequenceencoding a non-coding RNA molecule that targets at least one endogenousGA20 or GA3 oxidase gene for suppression, the transcribable DNA sequencebeing operably linked to a plant-expressible promoter, and wherein thetransgenic monocot or cereal plant has a shorter plant height relativeto a wild-type control plant.

In a ninth aspect, the present disclosure provides a cereal plantcomprising a mutation at or near an endogenous GA oxidase geneintroduced by a mutagenesis technique, wherein the expression level ofthe endogenous GA oxidase gene is reduced or eliminated in the cerealplant, and wherein the cereal plant has a shorter plant height relativeto a wild-type control plant.

In a tenth aspect, the present disclosure provides a corn or cerealplant comprising a genomic edit introduced via a targeted genome editingtechnique at or near the locus of an endogenous GA oxidase gene, whereinthe expression level of the endogenous GA oxidase gene is reduced oreliminated relative to a control plant, and wherein the edited cerealplant has a shorter plant height relative to the control plant.

In an eleventh aspect, the present disclosure provides a compositioncomprising a guide RNA, wherein the guide RNA comprises a guide sequencethat is at least 95%, at least 96%, at least 97%, at least 99%, or 100%identical or complementary to at least 10, at least 11, at least 12, atleast 13, at least 14, at least 15, at least 16, at least 17, at least18, at least 19, at least 20, at least 21, at least 22, at least 23, atleast 24, or at least 25 consecutive nucleotides of a target DNAsequence at or near the genomic locus of an endogenous GA oxidase geneof a cereal plant. In one aspect, a composition further comprises anRNA-guided endonuclease.

In a twelfth aspect, the present disclosure provides a recombinant DNAconstruct comprising a transcribable DNA sequence encoding a non-codingguide RNA molecule, wherein the guide RNA molecule comprises a guidesequence that is at least 95%, at least 96%, at least 97%, at least 99%or 100% complementary to at least 10, at least 11, at least 12, at least13, at least 14, at least 15, at least 16, at least 17, at least 18, atleast 19, at least 20, at least 21, at least 22, at least 23, at least24, or at least 25 consecutive nucleotides of a target DNA sequence ator near the genomic locus of an endogenous GA oxidase gene of a corn orcereal plant.

In a thirteenth aspect, the present disclosure provides a recombinantDNA donor template comprising at least one homology sequence, whereinthe at least one homology sequence is at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 99% or 100% complementary to at least 20, at least25, at least 30, at least 35, at least 40, at least 45, at least 50, atleast 60, at least 70, at least 80, at least 90, at least 100, at least150, at least 200, at least 250, at least 500, at least 1000, at least2500, or at least 5000 consecutive nucleotides of a target DNA sequence,wherein the target DNA sequence is a genomic sequence at or near thegenomic locus of an endogenous GA oxidase gene of a corn or cerealplant.

In a fourteenth aspect, the present disclosure provides a recombinantDNA donor template comprising two homology arms including a firsthomology arm and a second homology arm, wherein the first homology armcomprises a sequence that is at least 70%, at least 75%, at least 80%,at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 99% or 100% complementary to at least 20, at least 25, at least30, at least 35, at least 40, at least 45, at least 50, at least 60, atleast 70, at least 80, at least 90, at least 100, at least 150, at least200, at least 250, at least 500, at least 1000, at least 2500, or atleast 5000 consecutive nucleotides of a first flanking DNA sequence,wherein the second homology arm comprises a sequence that is at least70%, at least 75%, at least 80%, at least 85%, at least 90%, at least95%, at least 96%, at least 97%, at least 99% or 100% complementary toat least 20, at least 25, at least 30, at least 35, at least 40, atleast 45, at least 50, at least 60, at least 70, at least 80, at least90, at least 100, at least 150, at least 200, at least 250, at least500, at least 1000, at least 2500, or at least 5000 consecutivenucleotides of a second flanking DNA sequence, and wherein the firstflanking DNA sequence and the second flanking DNA sequence are genomicsequences at or near the genomic locus of an endogenous GA oxidase geneof a corn or cereal plant. In one aspect, further provided is a DNAmolecule or vector comprising a recombinant DNA donor template disclosedhere. In another aspect, further provided is a bacterial or host cellcomprising a recombinant DNA donor template disclosed here. In anotheraspect, further provided is corn or cereal plant, plant part or plantcell comprising the recombinant DNA construct disclosed here.

In a fifteenth aspect, the present disclosure provides an engineeredsite-specific nuclease that binds to a target site at or near thegenomic locus of an endogenous GA oxidase gene of a corn or cereal plantand causes a double-strand break or nick at the target site.

In a sixteenth aspect, the present disclosure provides a recombinant DNAconstruct comprising a transgene encoding a site-specific nuclease,wherein the site-specific nuclease binds to a target site at or near thegenomic locus of an endogenous GA oxidase gene of a monocot or cerealplant and causes a double-strand break or nick at the target site.

In a seventeenth aspect, the present disclosure provides a method forproducing a transgenic corn or cereal plant, comprising: (a)transforming at least one cell of an explant with a recombinant DNAdonor template disclosed here, and (b) regenerating or developing thetransgenic corn or cereal plant from the transformed explant, whereinthe transgenic corn or cereal plant comprises the insertion sequence ofthe recombinant DNA donor template.

In an eighteenth aspect, the present disclosure provides a method forproducing a corn or cereal plant having a genomic edit at or near anendogenous GA oxidase gene, comprising: (a) introducing into at leastone cell of an explant of the corn or cereal plant a site-specificnuclease or a recombinant DNA molecule comprising a transgene encodingthe site-specific nuclease, wherein the site-specific nuclease binds toa target site at or near the genomic locus of the endogenous GA oxidasegene and causes a double-strand break or nick at the target site, and(b) regenerating or developing an edited corn or cereal plant from theat least one explant cell comprising the genomic edit at or near theendogenous GA oxidase gene of the edited monocot or cereal plant.

In a nineteenth aspect, the present disclosure provides a modified cornplant having a plant height of less than 2000 mm, less than 1950 mm,less than 1900 mm, less than 1850 mm, less than 1800 mm, less than 1750mm, less than 1700 mm, less than 1650 mm, less than 1600 mm, less than1550 mm, less than 1500 mm, less than 1450 mm, less than 1400 mm, lessthan 1350 mm, less than 1300 mm, less than 1250 mm, less than 1200 mm,less than 1150 mm, less than 1100 mm, less than 1050 mm, or less than1000 mm, and one or more of (i) an average stem or stalk diameter ofgreater than 18 mm, greater than 18.5 mm, greater than 19 mm, greaterthan 19.5 mm, greater than 20 mm, greater than 20.5 mm, greater than 21mm, greater than 21.5 mm, or greater than 22 mm, (ii) improved lodgingresistance relative to a wild type control plant, or (iii) improveddrought tolerance relative to a wild type control plant.

In a twentieth aspect, the present disclosure provides a modified cerealplant having a reduced plant height relative to a wild type controlplant, and (i) an increased stem or stalk diameter relative to a wildtype control plant, (ii) improved lodging resistance relative to a wildtype control plant, or (iii) improved drought tolerance relative to awild type control plant.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows reduced plant heights of corn inbred plants expressing aGA20 oxidase suppression construct across eight transformation events incomparison to inbred control plants;

FIG. 2A shows a reduced plant height on average of hybrid corn plantsexpressing a GA20 oxidase suppression construct in comparison to hybridcontrol plants;

FIG. 2B shows an image of a wild type hybrid control plant (left) nextto a hybrid corn plant expressing a GA20 oxidase suppression construct(right) having a reduced plant height;

FIG. 3A shows an increased stem diameter on average of hybrid cornplants expressing a GA20 oxidase suppression construct in comparison tohybrid control plants;

FIG. 3B shows an image of a cross-section of the stalk of a wild typehybrid control plant (left) next to a cross-section of the stalk of ahybrid corn plant expressing a GA20 oxidase suppression construct(right) having an increased stem diameter;

FIG. 4 shows an increased fresh ear weight on average of hybrid cornplants expressing a GA20 oxidase suppression construct in comparison tohybrid control plants;

FIG. 5 shows the increased fresh ear weight on average of hybrid cornplants expressing a GA20 oxidase suppression construct in two fieldtrials in comparison to wild type hybrid control plants in response to awind event that caused greater lodging in the hybrid control plants;

FIG. 6 shows an increased harvest index of hybrid corn plants expressinga GA20 oxidase suppression construct in comparison to hybrid controlplants;

FIG. 7 shows an increase in the average grain yield estimate of hybridcorn plants expressing a GA20 oxidase suppression construct incomparison to hybrid control plants;

FIG. 8 shows an increased prolificacy score on average of hybrid cornplants expressing a GA20 oxidase suppression construct in comparison tohybrid control plants;

FIG. 9 shows the change in plant height over time during developmentalstages V11 to beyond R1 between transgenic corn plants and control;

FIG. 10 shows a graph comparing measurements of stable oxygen isotoperatios (δ¹⁸O) as an indication of stomatal conductance and water levelsin leaf tissue at R5 stage between transgenic corn plants and control;

FIG. 11 shows a graph comparing root front velocity during developmentalstages V10 to beyond R2 between transgenic and control plants at bothSAP and HD conditions using sensors at different soil depths that detectchanges in water levels indicating the presence of roots at that depth;

FIG. 12A shows differences in stomatal conductance during the morningand afternoon between transgenic corn plants and control under normaland drought conditions in the greenhouse;

FIG. 12B shows differences in photosynthesis during the morning andafternoon between transgenic corn plants and control under normal anddrought conditions in the greenhouse;

FIG. 13A shows differences in miRNA expression levels in bulk stemtissue, or separated vascular and non-vascular stem tissues, oftransgenic corn plants versus control; and

FIG. 13B shows differences in GA20 oxidase_3 and GA20 oxidase_5 mRNAtranscript expression levels in bulk stem tissue, or separated vascularand non-vascular stem tissues, of transgenic corn plants versus control.

DETAILED DESCRIPTION Definitions

To facilitate understanding of the disclosure, several terms andabbreviations as used herein are defined below as follows:

The term “and/or” when used in a list of two or more items, means thatany one of the listed items can be employed by itself or in combinationwith any one or more of the listed items. For example, the expression “Aand/or B” is intended to mean either or both of A and B—i.e., A alone, Balone, or A and B in combination. The expression “A, B and/or C” isintended to mean A alone, B alone, C alone, A and B in combination, Aand C in combination, B and C in combination, or A, B, and C incombination.

The term “about” as used herein, is intended to qualify the numericalvalues that it modifies, denoting such a value as variable within amargin of error. When no particular margin of error, such as a standarddeviation to a mean value, is recited, the term “about” should beunderstood to mean that range which would encompass the recited valueand the range which would be included by rounding up or down to thatfigure, taking into account significant figures.

The term “cereal plant” as used herein refers a monocotyledonous(monocot) crop plant that is in the Poaceae or Gramineae family ofgrasses and is typically harvested for its seed, including, for example,wheat, corn, rice, millet, barley, sorghum, oat and rye.

The terms “percent identity” or “percent identical” as used herein inreference to two or more nucleotide or protein sequences is calculatedby (i) comparing two optimally aligned sequences (nucleotide or protein)over a window of comparison, (ii) determining the number of positions atwhich the identical nucleic acid base (for nucleotide sequences) oramino acid residue (for proteins) occurs in both sequences to yield thenumber of matched positions, (iii) dividing the number of matchedpositions by the total number of positions in the window of comparison,and then (iv) multiplying this quotient by 100% to yield the percentidentity. For purposes of calculating “percent identity” between DNA andRNA sequences, a uracil (U) of a RNA sequence is considered identical toa thymine (T) of a DNA sequence. If the window of comparison is definedas a region of alignment between two or more sequences (i.e., excludingnucleotides at the 5′ and 3′ ends of aligned polynucleotide sequences,or amino acids at the N-terminus and C-terminus of aligned proteinsequences, that are not identical between the compared sequences), thenthe “percent identity” may also be referred to as a “percent alignmentidentity”. If the “percent identity” is being calculated in relation toa reference sequence without a particular comparison window beingspecified, then the percent identity is determined by dividing thenumber of matched positions over the region of alignment by the totallength of the reference sequence. Accordingly, for purposes of thepresent disclosure, when two sequences (query and subject) are optimallyaligned (with allowance for gaps in their alignment), the “percentidentity” for the query sequence is equal to the number of identicalpositions between the two sequences divided by the total number ofpositions in the query sequence over its length (or a comparisonwindow), which is then multiplied by 100%.

It is recognized that residue positions of proteins that are notidentical often differ by conservative amino acid substitutions, whereamino acid residues are substituted for other amino acid residues withsimilar size and chemical properties (e.g., charge, hydrophobicity,polarity, etc.), and therefore may not change the functional propertiesof the molecule. When sequences differ in conservative substitutions,the percent sequence similarity may be adjusted upwards to correct forthe conservative nature of the non-identical substitution(s). Sequencesthat differ by such conservative substitutions are said to have“sequence similarity” or “similarity.” Thus, “percent similarity” or“percent similar” as used herein in reference to two or more proteinsequences is calculated by (i) comparing two optimally aligned proteinsequences over a window of comparison, (ii) determining the number ofpositions at which the same or similar amino acid residue occurs in bothsequences to yield the number of matched positions, (iii) dividing thenumber of matched positions by the total number of positions in thewindow of comparison (or the total length of the reference or queryprotein if a window of comparison is not specified), and then (iv)multiplying this quotient by 100% to yield the percent similarity.Conservative amino acid substitutions for proteins are known in the art.

For optimal alignment of sequences to calculate their percent identityor similarity, various pair-wise or multiple sequence alignmentalgorithms and programs are known in the art, such as ClustalW, or BasicLocal Alignment Search Tool® (BLAST®), etc., that may be used to comparethe sequence identity or similarity between two or more nucleotide orprotein sequences. Although other alignment and comparison methods areknown in the art, the alignment between two sequences (including thepercent identity ranges described above) may be as determined by theClustalW or BLAST® algorithm, see, e.g., Chenna R. et al., “Multiplesequence alignment with the Clustal series of programs,” Nucleic AcidsResearch 31: 3497-3500 (2003); Thompson J D et al., “Clustal W:Improving the sensitivity of progressive multiple sequence alignmentthrough sequence weighting, position-specific gap penalties and weightmatrix choice,” Nucleic Acids Research 22: 4673-4680 (1994); and LarkinM A et al., “Clustal W and Clustal X version 2.0,” Bioinformatics 23:2947-48 (2007); and Altschul, S. F., Gish, W., Miller, W., Myers, E. W.& Lipman, D. J. (1990) “Basic local alignment search tool.” J. Mol.Biol. 215:403-410 (1990), the entire contents and disclosures of whichare incorporated herein by reference.

The terms “percent complementarity” or “percent complementary”, as usedherein in reference to two nucleotide sequences, is similar to theconcept of percent identity but refers to the percentage of nucleotidesof a query sequence that optimally base-pair or hybridize to nucleotidesof a subject sequence when the query and subject sequences are linearlyarranged and optimally base paired without secondary folding structures,such as loops, stems or hairpins. Such a percent complementarity may bebetween two DNA strands, two RNA strands, or a DNA strand and a RNAstrand. The “percent complementarity” is calculated by (i) optimallybase-pairing or hybridizing the two nucleotide sequences in a linear andfully extended arrangement (i.e., without folding or secondarystructures) over a window of comparison, (ii) determining the number ofpositions that base-pair between the two sequences over the window ofcomparison to yield the number of complementary positions, (iii)dividing the number of complementary positions by the total number ofpositions in the window of comparison, and (iv) multiplying thisquotient by 100% to yield the percent complementarity of the twosequences. Optimal base pairing of two sequences may be determined basedon the known pairings of nucleotide bases, such as G-C, A-T, and A-U,through hydrogen bonding. If the “percent complementarity” is beingcalculated in relation to a reference sequence without specifying aparticular comparison window, then the percent identity is determined bydividing the number of complementary positions between the two linearsequences by the total length of the reference sequence. Thus, forpurposes of the present disclosure, when two sequences (query andsubject) are optimally base-paired (with allowance for mismatches ornon-base-paired nucleotides but without folding or secondarystructures), the “percent complementarity” for the query sequence isequal to the number of base-paired positions between the two sequencesdivided by the total number of positions in the query sequence over itslength (or by the number of positions in the query sequence over acomparison window), which is then multiplied by 100%.

The term “operably linked” refers to a functional linkage between apromoter or other regulatory element and an associated transcribable DNAsequence or coding sequence of a gene (or transgene), such that thepromoter, etc., operates or functions to initiate, assist, affect,cause, and/or promote the transcription and expression of the associatedtranscribable DNA sequence or coding sequence, at least in certaincell(s), tissue(s), developmental stage(s), and/or condition(s).

The term “plant-expressible promoter” refers to a promoter that caninitiate, assist, affect, cause, and/or promote the transcription andexpression of its associated transcribable DNA sequence, coding sequenceor gene in a plant cell or tissue.

The term “heterologous” in reference to a promoter or other regulatorysequence in relation to an associated polynucleotide sequence (e.g., atranscribable DNA sequence or coding sequence or gene) is a promoter orregulatory sequence that is not operably linked to such associatedpolynucleotide sequence in nature—e.g., the promoter or regulatorysequence has a different origin relative to the associatedpolynucleotide sequence and/or the promoter or regulatory sequence isnot naturally occurring in a plant species to be transformed with thepromoter or regulatory sequence.

The term “recombinant” in reference to a polynucleotide (DNA or RNA)molecule, protein, construct, vector, etc., refers to a polynucleotideor protein molecule or sequence that is man-made and not normally foundin nature, and/or is present in a context in which it is not normallyfound in nature, including a polynucleotide (DNA or RNA) molecule,protein, construct, etc., comprising a combination of two or morepolynucleotide or protein sequences that would not naturally occurtogether in the same manner without human intervention, such as apolynucleotide molecule, protein, construct, etc., comprising at leasttwo polynucleotide or protein sequences that are operably linked butheterologous with respect to each other. For example, the term“recombinant” can refer to any combination of two or more DNA or proteinsequences in the same molecule (e.g., a plasmid, construct, vector,chromosome, protein, etc.) where such a combination is man-made and notnormally found in nature. As used in this definition, the phrase “notnormally found in nature” means not found in nature without humanintroduction. A recombinant polynucleotide or protein molecule,construct, etc., may comprise polynucleotide or protein sequence(s) thatis/are (i) separated from other polynucleotide or protein sequence(s)that exist in proximity to each other in nature, and/or (ii) adjacent to(or contiguous with) other polynucleotide or protein sequence(s) thatare not naturally in proximity with each other. Such a recombinantpolynucleotide molecule, protein, construct, etc., may also refer to apolynucleotide or protein molecule or sequence that has been geneticallyengineered and/or constructed outside of a cell. For example, arecombinant DNA molecule may comprise any engineered or man-madeplasmid, vector, etc., and may include a linear or circular DNAmolecule. Such plasmids, vectors, etc., may contain various maintenanceelements including a prokaryotic origin of replication and selectablemarker, as well as one or more transgenes or expression cassettesperhaps in addition to a plant selectable marker gene, etc.

As used herein, the term “isolated” refers to at least partiallyseparating a molecule from other molecules typically associated with itin its natural state. In one embodiment, the term “isolated” refers to aDNA molecule that is separated from the nucleic acids that normallyflank the DNA molecule in its natural state. For example, a DNA moleculeencoding a protein that is naturally present in a bacterium would be anisolated DNA molecule if it was not within the DNA of the bacterium fromwhich the DNA molecule encoding the protein is naturally found. Thus, aDNA molecule fused to or operably linked to one or more other DNAmolecule(s) with which it would not be associated in nature, for exampleas the result of recombinant DNA or plant transformation techniques, isconsidered isolated herein. Such molecules are considered isolated evenwhen integrated into the chromosome of a host cell or present in anucleic acid solution with other DNA molecules.

As used herein, an “encoding region” or “coding region” refers to aportion of a polynucleotide that encodes a functional unit or molecule(e.g., without being limiting, a mRNA, protein, or non-coding RNAsequence or molecule).

As used herein, “modified” in the context of a plant, plant seed, plantpart, plant cell, and/or plant genome, refers to a plant, plant seed,plant part, plant cell, and/or plant genome comprising an engineeredchange in the expression level and/or coding sequence of one or more GAoxidase gene(s) relative to a wild-type or control plant, plant seed,plant part, plant cell, and/or plant genome, such as via (A) atransgenic event comprising a suppression construct or transcribable DNAsequence encoding a non-coding RNA that targets one or more GA3 and/orGA20 oxidase genes for suppression, or (B) a genome editing event ormutation affecting (e.g., reducing or eliminating) the expression levelor activity of one or more endogenous GA3 and/or GA20 oxidase genes.Indeed, the term “modified” may further refer to a plant, plant seed,plant part, plant cell, and/or plant genome having one or more mutationsaffecting expression of one or more endogenous GA oxidase genes, such asone or more endogenous GA3 and/or GA20 oxidase genes, introduced throughchemical mutagenesis, transposon insertion or excision, or any otherknown mutagenesis technique, or introduced through genome editing. Forclarity, therefore, a modified plant, plant seed, plant part, plantcell, and/or plant genome includes a mutated, edited and/or transgenicplant, plant seed, plant part, plant cell, and/or plant genome having amodified expression level, expression pattern, and/or coding sequence ofone or more GA oxidase gene(s) relative to a wild-type or control plant,plant seed, plant part, plant cell, and/or plant genome. Modified plantsor seeds may contain various molecular changes that affect expression ofGA oxidase gene(s), such as GA3 and/or GA20 oxidase gene(s), includinggenetic and/or epigenetic modifications. Modified plants, plant parts,seeds, etc., may have been subjected to mutagenesis, genome editing orsite-directed integration (e.g., without being limiting, via methodsusing site-specific nucleases), genetic transformation (e.g., withoutbeing limiting, via methods of Agrobacterium transformation ormicroprojectile bombardment), or a combination thereof. Such “modified”plants, plant seeds, plant parts, and plant cells include plants, plantseeds, plant parts, and plant cells that are offspring or derived from“modified” plants, plant seeds, plant parts, and plant cells that retainthe molecular change (e.g., change in expression level and/or activity)to the one or more GA oxidase genes. A modified seed provided herein maygive rise to a modified plant provided herein. A modified plant, plantseed, plant part, plant cell, or plant genome provided herein maycomprise a recombinant DNA construct or vector or genome edit asprovided herein. A “modified plant product” may be any product made froma modified plant, plant part, plant cell, or plant chromosome providedherein, or any portion or component thereof.

As used herein, the term “control plant” (or likewise a “control” plantseed, plant part, plant cell and/or plant genome) refers to a plant (orplant seed, plant part, plant cell and/or plant genome) that is used forcomparison to a modified plant (or modified plant seed, plant part,plant cell and/or plant genome) and has the same or similar geneticbackground (e.g., same parental lines, hybrid cross, inbred line,testers, etc.) as the modified plant (or plant seed, plant part, plantcell and/or plant genome), except for a transgenic and/or genome editingevent(s) affecting one or more GA oxidase genes. For example, a controlplant may be an inbred line that is the same as the inbred line used tomake the modified plant, or a control plant may be the product of thesame hybrid cross of inbred parental lines as the modified plant, exceptfor the absence in the control plant of any transgenic or genome editingevent(s) affecting one or more GA oxidase genes. For purposes ofcomparison to a modified plant, plant seed, plant part, plant celland/or plant genome, a “wild-type plant” (or likewise a “wild-type”plant seed, plant part, plant cell and/or plant genome) refers to anon-transgenic and non-genome edited control plant, plant seed, plantpart, plant cell and/or plant genome. As used herein, a “control” plant,plant seed, plant part, plant cell and/or plant genome may also be aplant, plant seed, plant part, plant cell and/or plant genome having asimilar (but not the same or identical) genetic background to a modifiedplant, plant seed, plant part, plant cell and/or plant genome, if deemedsufficiently similar for comparison of the characteristics or traits tobe analyzed.

As used herein, a “target site” for genome editing refers to thelocation of a polynucleotide sequence within a plant genome that isbound and cleaved by a site-specific nuclease introducing a doublestranded break (or single-stranded nick) into the nucleic acid backboneof the polynucleotide sequence and/or its complementary DNA strand. Atarget site may comprise at least 10, at least 11, at least 12, at least13, at least 14, at least 15, at least 16, at least 17, at least 18, atleast 19, at least 20, at least 21, at least 22, at least 23, at least24, at least 25, at least 26, at least 27, at least 29, or at least 30consecutive nucleotides. A “target site” for a RNA-guided nuclease maycomprise the sequence of either complementary strand of adouble-stranded nucleic acid (DNA) molecule or chromosome at the targetsite. A site-specific nuclease may bind to a target site, such as via anon-coding guide RNA (e.g., without being limiting, a CRISPR RNA (crRNA)or a single-guide RNA (sgRNA) as described further below). A non-codingguide RNA provided herein may be complementary to a target site (e.g.,complementary to either strand of a double-stranded nucleic acidmolecule or chromosome at the target site). It will be appreciated thatperfect identity or complementarity may not be required for a non-codingguide RNA to bind or hybridize to a target site. For example, at least1, at least 2, at least 3, at least 4, at least 5, at least 6, at least7, or at least 8 mismatches (or more) between a target site and anon-coding RNA may be tolerated. A “target site” also refers to thelocation of a polynucleotide sequence within a plant genome that isbound and cleaved by another site-specific nuclease that may not beguided by a non-coding RNA molecule, such as a meganuclease, zinc fingernuclease (ZFN), or a transcription activator-like effector nuclease(TALEN), to introduce a double stranded break (or single-stranded nick)into the polynucleotide sequence and/or its complementary DNA strand. Asused herein, a “target region” or a “targeted region” refers to apolynucleotide sequence or region that is flanked by two or more targetsites. Without being limiting, in some embodiments a target region maybe subjected to a mutation, deletion, insertion or inversion. As usedherein, “flanked” when used to describe a target region of apolynucleotide sequence or molecule, refers to two or more target sitesof the polynucleotide sequence or molecule surrounding the targetregion, with one target site on each side of the target region. Apartfrom genome editing, the term “target site” may also be used in thecontext of gene suppression to refer to a portion of a mRNA molecule(e.g., a “recognition site”) that is complementary to at least a portionof a non-coding RNA molecule (e.g., a miRNA, siRNA, etc.) encoded by asuppression construct.

As used herein, a “donor molecule”, “donor template”, or “donor templatemolecule” (collectively a “donor template”), which may be a recombinantDNA donor template, is defined as a nucleic acid molecule having anucleic acid template or insertion sequence for site-directed, targetedinsertion or recombination into the genome of a plant cell via repair ofa nick or double-stranded DNA break in the genome of a plant cell. Forexample, a “donor template” may be used for site-directed integration ofa transgene or suppression construct, or as a template to introduce amutation, such as an insertion, deletion, etc., into a target sitewithin the genome of a plant. A targeted genome editing techniqueprovided herein may comprise the use of one or more, two or more, threeor more, four or more, or five or more donor molecules or templates. A“donor template” may be a single-stranded or double-stranded DNA or RNAmolecule or plasmid. An “insertion sequence” of a donor template is asequence designed for targeted insertion into the genome of a plantcell, which may be of any suitable length. For example, the insertionsequence of a donor template may be between 2 and 50,000, between 2 and10,000, between 2 and 5000, between 2 and 1000, between 2 and 500,between 2 and 250, between 2 and 100, between 2 and 50, between 2 and30, between 15 and 50, between 15 and 100, between 15 and 500, between15 and 1000, between 15 and 5000, between 18 and 30, between 18 and 26,between 20 and 26, between 20 and 50, between 20 and 100, between 20 and250, between 20 and 500, between 20 and 1000, between 20 and 5000,between 20 and 10,000, between 50 and 250, between 50 and 500, between50 and 1000, between 50 and 5000, between 50 and 10,000, between 100 and250, between 100 and 500, between 100 and 1000, between 100 and 5000,between 100 and 10,000, between 250 and 500, between 250 and 1000,between 250 and 5000, or between 250 and 10,000 nucleotides or basepairs in length. A donor template may also have at least one homologysequence or homology arm, such as two homology arms, to direct theintegration of a mutation or insertion sequence into a target sitewithin the genome of a plant via homologous recombination, wherein thehomology sequence or homology arm(s) are identical or complementary, orhave a percent identity or percent complementarity, to a sequence at ornear the target site within the genome of the plant. When a donortemplate comprises homology arm(s) and an insertion sequence, thehomology arm(s) will flank or surround the insertion sequence of thedonor template.

An insertion sequence of a donor template may comprise one or more genesor sequences that each encode a transcribed non-coding RNA or mRNAsequence and/or a translated protein sequence. A transcribed sequence orgene of a donor template may encode a protein or a non-coding RNAmolecule. An insertion sequence of a donor template may comprise apolynucleotide sequence that does not comprise a functional gene or anentire gene sequence (e.g., the donor template may simply compriseregulatory sequences, such as a promoter sequence, or only a portion ofa gene or coding sequence), or may not contain any identifiable geneexpression elements or any actively transcribed gene sequence. Further,the donor template may be linear or circular, and may be single-strandedor double-stranded. A donor template may be delivered to the cell as anaked nucleic acid (e.g., via particle bombardment), as a complex withone or more delivery agents (e.g., liposomes, proteins, poloxamers,T-strand encapsulated with proteins, etc.), or contained in a bacterialor viral delivery vehicle, such as, for example, Agrobacteriumtumefaciens or a geminivirus, respectively. An insertion sequence of adonor template provided herein may comprise a transcribable DNA sequencethat may be transcribed into an RNA molecule, which may be non-codingand may or may not be operably linked to a promoter and/or otherregulatory sequence.

According to some embodiments, a donor template may not comprise aninsertion sequence, and instead comprise one or more homology sequencesthat include(s) one or more mutations, such as an insertion, deletion,substitution, etc., relative to the genomic sequence at a target sitewithin the genome of a plant, such as at or near a GA3 oxidase or GA20oxidase gene within the genome of a plant. Alternatively, a donortemplate may comprise an insertion sequence that does not comprise acoding or transcribable DNA sequence, wherein the insertion sequence isused to introduce one or more mutations into a target site within thegenome of a plant, such as at or near a GA3 oxidase or GA20 oxidase genewithin the genome of a plant.

A donor template provided herein may comprise at least one, at leasttwo, at least three, at least four, at least five, at least six, atleast seven, at least eight, at least nine, or at least ten genes ortranscribable DNA sequences. Alternatively, a donor template maycomprise no genes. Without being limiting, a gene or transcribable DNAsequence of a donor template may include, for example, an insecticidalresistance gene, an herbicide tolerance gene, a nitrogen use efficiencygene, a water use efficiency gene, a nutritional quality gene, a DNAbinding gene, a selectable marker gene, an RNAi or suppressionconstruct, a site-specific genome modification enzyme gene, a singleguide RNA of a CRISPR/Cas9 system, a geminivirus-based expressioncassette, or a plant viral expression vector system. According to otherembodiments, an insertion sequence of a donor template may comprise atranscribable DNA sequence that encodes a non-coding RNA molecule, whichmay target a GA oxidase gene, such as a GA3 oxidase or GA20 oxidasegene, for suppression. A donor template may comprise a promoter, such asa tissue-specific or tissue-preferred promoter, a constitutive promoter,or an inducible promoter. A donor template may comprise a leader,enhancer, promoter, transcriptional start site, 5′-UTR, one or moreexon(s), one or more intron(s), transcriptional termination site, regionor sequence, 3′-UTR, and/or polyadenylation signal. The leader,enhancer, and/or promoter may be operably linked to a gene ortranscribable DNA sequence encoding a non-coding RNA, a guide RNA, anmRNA and/or protein.

As used herein, a “vascular promoter” refers to a plant-expressiblepromoter that drives, causes or initiates expression of a transcribableDNA sequence or transgene operably linked to such promoter in one ormore vascular tissue(s) of the plant, even if the promoter is alsoexpressed in other non-vascular plant cell(s) or tissue(s). Suchvascular tissue(s) may comprise one or more of the phloem, vascularparenchymal, and/or bundle sheath cell(s) or tissue(s) of the plant. A“vascular promoter” is distinguished from a constitutive promoter inthat it has a regulated and relatively more limited pattern ofexpression that includes one or more vascular tissue(s) of the plant. Avascular promoter includes both vascular-specific promoters andvascular-preferred promoters.

As used herein, a “leaf promoter” refers to a plant-expressible promoterthat drives, causes or initiates expression of a transcribable DNAsequence or transgene operably linked to such promoter in one or moreleaf tissue(s) of the plant, even if the promoter is also expressed inother non-leaf plant cell(s) or tissue(s). A leaf promoter includes bothleaf-specific promoters and leaf-preferred promoters. A “leaf promoter”is distinguished from a vascular promoter in that it is expressed morepredominantly or exclusively in leaf tissue(s) of the plant relative toother plant tissues, whereas a vascular promoter is expressed invascular tissue(s) more generally including vascular tissue(s) outsideof the leaf, such as the vascular tissue(s) of the stem, or stem andleaves, of the plant.

As used herein, a “plant-expressible promoter” refers to a promoter thatdrives, causes or initiates expression of a transcribable DNA sequenceor transgene operably linked to such promoter in one or more plant cellsor tissues, such as one or more cells or tissues of a corn or cerealplant.

DESCRIPTION

Most grain producing grasses, such as wheat, rice and sorghum, produceboth male and female structures within each floret of the panicle (i.e.,they have a single reproductive structure). However, corn or maize isunique among the grain-producing grasses in that it forms separate male(tassel) and female (ear) inflorescences. Corn produces completelysexually dimorphic reproductive structures by selective abortion of maleorgans (anthers) in florets of the ear, and female organs (ovules) inthe florets of the tassel within early stages of development. Preciselyregulated gibberellin synthesis and signaling is critical to regulationof this selective abortion process, with the female reproductive earbeing most sensitive to disruptions in the GA pathway. Indeed, the“anther ear” phenotype is the most common reproductive phenotype in GAcorn mutants.

In contrast to corn, mutations in the gibberellin synthesis or signalingpathways that led to the “Green Revolution” in wheat, rice and sorghumhad little impact on their reproductive structures because these cropspecies do not undergo the selective abortion process of the grainbearing panicle during development, and thus are not sensitive todisruptions in GA levels. The same mutations have not been utilized incorn because disruption of the GA synthesis and signaling pathway hasrepeatedly led to dramatic distortion and masculinization of the ear(“anther ear”) and sterility (disrupted anther and microsporedevelopment) in the tassel, in addition to extreme dwarfing in somecases. See, e.g., Chen, Y. et al., “The Maize DWARF1 Encodes aGibberellin 3-Oxidase and Is Dual Localized to the Nucleus and Cytosol,”Plant Physiology 166: 2028-2039 (2014). These GA mutant phenotypes(off-types) in corn led to significant reductions in kernel productionand a reduction in yield. Furthermore, production of anthers within theear increases the likelihood of fungal or insect infections, whichreduces the quality of the grain that is produced on those mutant ears.Forward breeding to develop semi-dwarf lines of corn has not beensuccessful, and the reproductive off-types (as well as the extremedwarfing) of GA mutants have been challenging to overcome. Thus, thesame mutations in the GA pathway that led to the Green Revolution inother grasses have not yet been successful in corn.

Despite these prior difficulties in achieving higher grain yields incorn through manipulation of the GA pathway, the present inventors havediscovered a way to manipulate GA levels in corn plants in a manner thatreduces overall plant height and stem internode length and increasesresistance to lodging, but does not cause the reproductive off-typespreviously associated with mutations of the GA pathway in corn. Furtherevidence indicates that these short stature or semi-dwarf corn plantsmay also have one or more additional traits, including increased stemdiameter, reduced green snap, deeper roots, increased leaf area, earliercanopy closure, higher stomatal conductance, lower ear height, increasedfoliar water content, improved drought tolerance, increased nitrogen useefficiency, increased water use efficiency, reduced anthocyanin contentand area in leaves under normal or nitrogen or water limiting stressconditions, increased ear weight, increased kernel number, increasedkernel weight, increased yield, and/or increased harvest index.

Without being bound by theory, it is proposed that incompletesuppression of GA20 or GA3 oxidase gene(s) and/or targeting of a subsetof one or more GA oxidase gene(s) may be effective in achieving a shortstature, semi-dwarf phenotype with increased resistance to lodging, butwithout reproductive off-types in the ear. It is further proposed,without being limited by theory, that restricting the suppression ofGA20 and/or GA3 oxidase gene(s) to certain active GA-producing tissues,such as the vascular and/or leaf tissues of the plant, may be sufficientto produce a short-stature plant with increased lodging resistance, butwithout significant off-types in reproductive tissues. Expression of aGA20 or GA3 oxidase suppression element in a tissue-specific ortissue-preferred manner may be sufficient and effective at producingplants with the short stature phenotype, while avoiding potentialoff-types in reproductive tissues that were previously observed with GAmutants in corn (e.g., by avoiding or limiting the suppression of theGA20 oxidase gene(s) in those reproductive tissues). For example, GA20and/or GA3 oxidase gene(s) may be targeted for suppression using avascular promoter, such as a rice tungro bacilliform virus (RTBV)promoter, that drives expression in vascular tissues of plants. Assupported in the Examples below, the expression pattern of the RTBVpromoter is enriched in vascular tissues of corn plants relative tonon-vascular tissues, which is sufficient to produce a semi-dwarfphenotype in corn plants when operably linked to a suppression elementtargeting GA20 and GA3 oxidase gene(s). Lowering of active GA levels intissue(s) of a corn or cereal plant that produce active GAs may reduceplant height and increase lodging resistance, and off-types may beavoided in those plants if active GA levels are not also significantlyimpacted or lowered in reproductive tissues, such as the developingfemale organ or ear of the plant. If active GA levels could be reducedin the stalk, stem, or internode(s) of corn or cereal plants withoutsignificantly affecting GA levels in reproductive tissues (e.g., thefemale or male reproductive organs or inflorescences), then corn orcereal plants having reduced plant height and increased lodgingresistance could be created without off-types in the reproductivetissues of the plant.

Thus, recombinant DNA constructs and transgenic plants are providedherein comprising a GA20 or GA3 oxidase suppression element or sequenceoperably linked to a plant expressible promoter, which may be atissue-specific or tissue-preferred promoter. Such a tissue-specific ortissue-preferred promoter may drive expression of its associated GAoxidase suppression element or sequence in one or more activeGA-producing tissue(s) of the plant to suppress or reduce the level ofactive GAs produced in those tissue(s). Such a tissue-specific ortissue-preferred promoter may drive expression of its associated GAoxidase suppression construct or transgene during one or more vegetativestage(s) of development. Such a tissue-specific or tissue-preferredpromoter may also have little or no expression in one or more cell(s) ortissue(s) of the developing female organ or ear of the plant to avoidthe possibility of off-types in those reproductive tissues. According tosome embodiments, the tissue-specific or tissue-preferred promoter is avascular promoter, such as the RTBV promoter. The sequence of the RTBVpromoter is provided herein as SEQ ID NO: 65, and a truncated version ofthe RTBV promoter is further provided herein as SEQ ID NO: 66.

Active or bioactive gibberellic acids (i.e., “active gibberellins” or“active GAs”) are known in the art for a given plant species, asdistinguished from inactive GAs. For example, active GAs in corn andhigher plants include the following: GA1, GA3, GA4, and GA7. Thus, an“active GA-producing tissue” is a plant tissue that produces one or moreactive GAs.

In addition to suppressing GA20 oxidase genes in active GA-producingtissues of the plant with a vascular tissue promoter, it wassurprisingly found that suppression of the same GA20 oxidase genes withvarious constitutive promoters could also cause the short, semi-dwarfstature phenotypes in corn, but without any visible off-types in theear. Given that mutations in the GA pathway have previously been shownto cause off-types in reproductive tissues, it was surprising thatconstitutive suppression of GA20 oxidase did not cause similarreproductive phenotypes in the ear. Thus, it is further proposed thatsuppression of one or more GA20 oxidase genes could be carried out usinga constitutive promoter to create a short stature, lodging-resistantcorn or cereal plant without any significant or observable reproductiveoff-types in the plant. Other surprising observations were made when thesame GA20 oxidase suppression construct was expressed in the stem, leafor reproductive tissues. As described further below, targetedsuppression of the same GA20 oxidase genes in the stem or ear tissues ofcorn plants did not cause the short stature, semi-dwarf phenotype.Moreover, directed expression of the GA20 oxidase suppression constructdirectly in reproductive tissues of the developing ear of corn plantswith a female reproductive tissue (ear) promoter did not cause anysignificant or observable off-types in the ear. However, expression ofthe same GA20 oxidase suppression construct in leaf tissues wassufficient to cause a moderate short stature phenotype withoutsignificant or observable reproductive off-types in the plant.

Without being limited by theory, it is proposed that short stature,semi-dwarf phenotypes in corn and other cereal plants may result from asufficient level of expression of a suppression construct targetingcertain GA oxidase gene(s) in active GA-producing tissue(s) of theplant. At least for targeted suppression of certain GA20 oxidase genesin corn, restricting the pattern of expression to avoid reproductive eartissues may not be necessary to avoid reproductive off-types in thedeveloping ear. However, expression of the GA20 oxidase suppressionconstruct at low levels, and/or in a limited number of plant tissues,may be insufficient to cause a significant short stature, semi-dwarfphenotype. Given that the observed semi-dwarf phenotype with targetedGA20 oxidase suppression is the result of shortening the stem internodesof the plant, it is surprising that suppression of GA20 oxidase genes inat least some stem tissues was not sufficient to cause shortening of theinternodes and reduced plant height. Without being bound by theory, itis proposed that suppression of certain GA oxidase gene(s) in tissue(s)and/or cell(s) of the plant where active GAs are produced, and notnecessarily in stem or internode tissue(s), may be sufficient to producesemi-dwarf plants, even though the short stature trait is due toshortening of the stem internodes. Given that GAs can migrate throughthe vasculature of the plant, it is proposed that manipulating GAoxidase genes in plant tissue(s) where active GAs are produced mayresult in a short stature, semi-dwarf plant, even though this may belargely achieved by suppressing the level of active GAs produced innon-stem tissues (i.e., away from the site of action in the stem wherereduced internode elongation leads to the semi-dwarf phenotype). Indeed,suppression of certain GA20 oxidase genes in leaf tissues was found tocause a moderate semi-dwarf phenotype in corn plants. Given thatexpression of a GA20 oxidase suppression construct with severaldifferent “stem” promoters did not produce the semi-dwarf phenotype incorn, it is noteworthy that expression of the same GA20 oxidasesuppression construct with a vascular promoter was effective atconsistently producing the semi-dwarf phenotype with a high degree ofpenetrance across events and germplasms. This semi-dwarf phenotype wasalso observed with expression of the same GA20 oxidase suppressionconstruct using other vascular promoters.

According to embodiments of the present disclosure, modified cereal orcorn plants are provided that have at least one beneficial agronomictrait and at least one female reproductive organ or ear that issubstantially or completely free of off-types. The beneficial agronomictrait may include, for example, shorter plant height, shorter internodelength in one or more internode(s), larger (thicker) stem or stalkdiameter, increased lodging resistance, improved drought tolerance,increased nitrogen use efficiency, increased water use efficiency,deeper roots, larger leaf area, earlier canopy closure, and/or increasedharvestable yield. Off-types may include male (tassel or anther)sterility, reduced kernel or seed number, and/or the presence of one ormore masculinized or male (or male-like) reproductive structures in thefemale organ or ear (e.g., anther ear) of the plant. A modified cerealor corn plant is provided herein that lacks significant off-types in thereproductive tissues of the plant. Such a modified cereal or corn plantmay have a female reproductive organ or ear that appears normal relativeto a control or wild-type plant. Indeed, modified cereal or corn plantsare provided that comprise at least one reproductive organ or ear thatdoes not have or exhibit, or is substantially or completely free of,off-types including male sterility, reduced kernel or seed number,and/or masculinized structure(s) in one or more female organs or ears.As used herein, a female organ or ear of a plant, such as corn, is“substantially free” of male reproductive structures if malereproductive structures are absent or nearly absent in the female organor ear of the plant based on visual inspection of the female organ orear at later reproductive stages. A female organ or ear of a plant, suchas corn, is “completely free” of mature male reproductive structures ifmale reproductive structures are absent or not observed or observable inthe female organ or ear of the plant, such as a corn plant, by visualinspection of the female organ or ear at later reproductive stages. Afemale organ or ear of a plant, such as corn, without significantoff-types and substantially free of male reproductive structures in theear may have a number of kernels or seeds per female organ or ear of theplant that is at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%,at least 99.8%, or at least 99.9% of the number of kernels or seeds perfemale organ or ear of a wild-type or control plant. Likewise, a femaleorgan or ear of a plant, such as corn, without significant off-types andsubstantially free of male reproductive structures in the ear may havean average kernel or seed weight per female organ or ear of the plantthat is at least 90%, at least 95%, at least 96%, at least 97%, at least98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, atleast 99.8%, or at least 99.9% of the average kernel or seed weight perfemale organ or ear of a wild-type or control plant. A female organ orear of a plant, such as corn, that is completely free of mature malereproductive structures may have a number of kernels or seeds per femaleorgan or ear of the plant that is about the same as a wild-type orcontrol plant. In other words, the reproductive development of thefemale organ or ear of the plant may be normal or substantially normal.However, the number of seeds or kernels per female organ or ear maydepend on other factors that affect resource utilization and developmentof the plant. Indeed, the number of kernels or seeds per female organ orear of the plant, and/or the kernel or seed weight per female organ orear of the plant, may be about the same or greater than a wild-type orcontrol plant.

The plant hormone gibberellin plays an important role in a number ofplant developmental processes including germination, cell elongation,flowering, embryogenesis and seed development. Certain biosyntheticenzymes (e.g., GA20 oxidase and GA3 oxidase) and catabolic enzymes(e.g., GA2 oxidase) in the GA pathway are critical to affecting activeGA levels in plant tissues. Thus, in addition to suppression of certainGA20 oxidase genes, it is further proposed that suppression of a GA3oxidase gene in a constitutive or tissue-specific or tissue-preferredmanner may also produce corn plants having a short stature phenotype andincreased lodging resistance, with possible increased yield, but withoutoff-types in the ear. Thus, according to some embodiments, constructsand transgenes are provided comprising a GA3 oxidase suppression elementor sequence operably linked to a constitutive or tissue-specific ortissue-preferred promoter, such as a vascular or leaf promoter.According to some embodiments, the tissue-specific or tissue-preferredpromoter is a vascular promoter, such as the RTBV promoter. However,other types of tissue-specific or tissue preferred promoters maypotentially be used for GA3 oxidase suppression in active GA-producingtissues of a corn or cereal plant to produce a semi-dwarf phenotypewithout significant off-types.

Any method known in the art for suppression of a target gene may be usedto suppress GA oxidase gene(s) according to embodiments of the presentinvention including expression of antisense RNAs, double stranded RNAs(dsRNAs) or inverted repeat RNA sequences, or via co-suppression or RNAinterference (RNAi) through expression of small interfering RNAs(siRNAs), short hairpin RNAs (shRNAs), trans-acting siRNAs (ta-siRNAs),or micro RNAs (miRNAs). Furthermore, sense and/or antisense RNAmolecules may be used that target the coding and/or non-coding genomicsequences or regions within or near a GA oxidase gene to cause silencingof the gene. Accordingly, any of these methods may be used for thetargeted suppression of an endogenous GA20 oxidase(s) or GA3 oxidasegene(s) in a tissue-specific or tissue-preferred manner. See, e.g., U.S.Patent Application Publication Nos. 2009/0070898, 2011/0296555, and2011/0035839, the contents and disclosures of which are incorporatedherein by reference.

The term “suppression” as used herein, refers to a lowering, reductionor elimination of the expression level of a mRNA and/or protein encodedby a target gene in a plant, plant cell or plant tissue at one or morestage(s) of plant development, as compared to the expression level ofsuch target mRNA and/or protein in a wild-type or control plant, cell ortissue at the same stage(s) of plant development. According to someembodiments, a modified or transgenic plant is provided having a GA20oxidase gene expression level that is reduced in at least one planttissue by at least 5%, at least 10%, at least 20%, at least 25%, atleast 30%, at least 40%, at least 50%, at least 60%, at least 70%, atleast 75%, at least 80%, at least 90%, or 100%, as compared to a controlplant. According to some embodiments, a modified or transgenic plant isprovided having a GA3 oxidase gene expression level that is reduced inat least one plant tissue by at least 5%, at least 10%, at least 20%, atleast 25%, at least 30%, at least 40%, at least 50%, at least 60%, atleast 70%, at least 75%, at least 80%, at least 90%, or 100%, ascompared to a control plant. According to some embodiments, a modifiedor transgenic plant is provided having a GA20 oxidase gene expressionlevel that is reduced in at least one plant tissue by 5%-20%, 5%-25%,5%-30%, 5%-40%, 5%-50%, 5%-60%, 5%-70%, 5%-75%, 5%-80%, 5%-90%, 5%-100%,75%-100%, 50%-100%, 50%-90%, 50%-75%, 25%-75%, 30%-80%, or 10%-75%, ascompared to a control plant. According to some embodiments, a modifiedor transgenic plant is provided having a GA3 oxidase gene expressionlevel that is reduced in at least one plant tissue by 5%-20%, 5%-25%,5%-30%, 5%-40%, 5%-50%, 5%-60%, 5%-70%, 5%-75%, 5%-80%, 5%-90%, 5%-100%,75%-100%, 50%-100%, 50%-90%, 50%-75%, 25%-75%, 30%-80%, or 10%-75%, ascompared to a control plant. According to these embodiments, the atleast one tissue of a modified or transgenic plant having a reducedexpression level of a GA20 oxidase and/or GA3 oxidase gene(s) includesone or more active GA producing tissue(s) of the plant, such as thevascular and/or leaf tissue(s) of the plant, during one or morevegetative stage(s) of development.

In some embodiments, suppression of an endogenous GA20 oxidase gene or aGA3 oxidase gene is tissue-specific (e.g., only in leaf and/or vasculartissue). Suppression of a GA20 oxidase gene may be constitutive and/orvascular or leaf tissue specific or preferred. In other embodiments,suppression of a GA20 oxidase gene or a GA3 oxidase gene is constitutiveand not tissue-specific. According to some embodiments, expression of anendogenous GA20 oxidase gene and/or a GA3 oxidase gene is reduced in oneor more tissue types (e.g., in leaf and/or vascular tissue(s)) of amodified or transgenic plant as compared to the same tissue(s) of acontrol plant.

According to embodiments of the present disclosure, a recombinant DNAmolecule, construct or vector is provided comprising a suppressionelement targeting GA20 oxidase or GA3 oxidase gene(s) that is operablylinked to a plant-expressible constitutive or tissue-specific ortissue-preferred promoter. The suppression element may comprise atranscribable DNA sequence of at least 19 nucleotides in length, such asfrom about 19 nucleotides in length to about 27 nucleotides in length,or 19, 20, 21, 22, 23, 24, 25, 26, or 27 nucleotides in length, whereinthe transcribable DNA sequence corresponds to at least a portion of thetarget GA oxidase gene to be suppressed, and/or to a DNA sequencecomplementary thereto. The suppression element may be 19-30, 19-50,19-100, 19-200, 19-300, 19-500, 19-1000, 19-1500, 19-2000, 19-3000,19-4000, or 19-5000 nucleotides in length. The suppression element maybe at least 19, at least 20, at least 21, at least 22, or at least 23nucleotides or more in length (e.g., at least 25, at least 30, at least50, at least 100, at least 200, at least 300, at least 500, at least1000, at least 1500, at least 2000, at least 3000, at least 4000, or atleast 5000 nucleotides in length). Depending on the length and sequenceof a suppression element, one or more sequence mismatches ornon-complementary bases, such as 1, 2, 3, 4, 5, 6, 7, 8 or moremismatches, may be tolerated without a loss of suppression if thenon-coding RNA molecule encoded by the suppression element is still ableto sufficiently hybridize and bind to the target mRNA molecule of theGA20 oxidase or GA3 oxidase gene(s). Indeed, even shorter RNAisuppression elements ranging from about 19 nucleotides to about 27nucleotides in length may have one or more mismatches ornon-complementary bases, yet still be effective at suppressing a targetGA oxidase gene. Accordingly, a sense or anti-sense suppression elementsequence may be at least 80%, at least 85%, at least 90%, at least 95%,at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%or 100% identical to a corresponding sequence of at least a segment orportion of the targeted GA oxidase gene, or its complementary sequence,respectively.

A suppression element or transcribable DNA sequence of the presentinvention for targeted suppression of GA oxidase gene(s) may include oneor more of the following: (a) a DNA sequence that includes at least oneanti-sense DNA sequence that is anti-sense or complementary to at leastone segment or portion of the targeted GA oxidase gene; (b) a DNAsequence that includes multiple copies of at least one anti-sense DNAsequence that is anti-sense or complementary to at least one segment orportion of the targeted GA oxidase gene; (c) a DNA sequence thatincludes at least one sense DNA sequence that comprises at least onesegment or portion of the targeted GA oxidase gene; (d) a DNA sequencethat includes multiple copies of at least one sense DNA sequence thateach comprise at least one segment or portion of the targeted GA oxidasegene; (e) a DNA sequence that includes an inverted repeat of a segmentor portion of a targeted GA oxidase gene and/or transcribes into RNA forsuppressing the targeted GA oxidase gene by forming double-stranded RNA,wherein the transcribed RNA includes at least one anti-sense DNAsequence that is anti-sense or complementary to at least one segment orportion of the targeted GA oxidase gene and at least one sense DNAsequence that comprises at least one segment or portion of the targetedGA oxidase gene; (f) a DNA sequence that is transcribed into RNA forsuppressing the targeted GA oxidase gene by forming a singledouble-stranded RNA and includes multiple serial anti-sense DNAsequences that are each anti-sense or complementary to at least onesegment or portion of the targeted GA oxidase gene and multiple serialsense DNA sequences that each comprise at least one segment or portionof the targeted GA oxidase gene; (g) a DNA sequence that is transcribedinto RNA for suppressing the targeted GA oxidase gene by formingmultiple double strands of RNA and includes multiple anti-sense DNAsequences that are each anti-sense or complementary to at least onesegment or portion of the targeted GA oxidase gene and multiple senseDNA sequences that each comprise at least one segment or portion of thetargeted GA oxidase gene, wherein the multiple anti-sense DNA segmentsand multiple sense DNA segments are arranged in a series of invertedrepeats; (h) a DNA sequence that includes nucleotides derived from amiRNA, preferably a plant miRNA; (i) a DNA sequence that includes amiRNA precursor that encodes an artificial miRNA complementary to atleast one segment or portion of the targeted GA oxidase gene; (j) a DNAsequence that includes nucleotides of a siRNA; (k) a DNA sequence thatis transcribed into an RNA aptamer capable of binding to a ligand; and(l) a DNA sequence that is transcribed into an RNA aptamer capable ofbinding to a ligand and DNA that transcribes into a regulatory RNAcapable of regulating expression of the targeted GA oxidase gene,wherein the regulation of the targeted GA oxidase gene is dependent onthe conformation of the regulatory RNA, and the conformation of theregulatory RNA is allosterically affected by the binding state of theRNA aptamer by the ligand. Any of these gene suppression elements,whether transcribed into a single stranded or double-stranded RNA, maybe designed to suppress more than one GA oxidase target gene, dependingon the number and sequence of the suppression element(s).

Multiple sense and/or anti-sense suppression elements for more than oneGA oxidase target may be arranged serially in tandem or arranged intandem segments or repeats, such as tandem inverted repeats, which mayalso be interrupted by one or more spacer sequence(s), and the sequenceof each suppression element may target one or more GA oxidase gene(s).Furthermore, the sense or anti-sense sequence of the suppression elementmay not be perfectly matched or complementary to the targeted GA oxidasegene sequence, depending on the sequence and length of the suppressionelement. Even shorter RNAi suppression elements from about 19nucleotides to about 27 nucleotides in length may have one or moremismatches or non-complementary bases, yet still be effective atsuppressing the target GA oxidase gene. Accordingly, a sense oranti-sense suppression element sequence may be at least 80%, at least85%, at least 90%, at least 95%, at least 96%, at least 97%, at least98%, at least 99%, at least 99.5% or 100% identical to a correspondingsequence of at least a segment or portion of the targeted GA oxidasegene, or its complementary sequence, respectively.

For anti-sense suppression, the transcribable DNA sequence orsuppression element comprises a sequence that is anti-sense orcomplementary to at least a portion or segment of the targeted GAoxidase gene. The suppression element may comprise multiple anti-sensesequences that are complementary to one or more portions or segments ofthe targeted GA oxidase gene(s), or multiple copies of an anti-sensesequence that is complementary to a targeted GA oxidase gene. Theanti-sense suppression element sequence may be at least 80%, at least85%, at least 90%, at least 95%, at least 96%, at least 97%, at least98%, at least 99%, at least 99.5% or 100% identical to a DNA sequencethat is complementary to at least a segment or portion of the targetedGA oxidase gene. In other words, the anti-sense suppression elementsequence may be at least 80%, at least 85%, at least 90%, at least 95%,at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%or 100% complementary to the targeted GA oxidase gene.

For suppression of GA oxidase gene(s) using an inverted repeat or atranscribed dsRNA, a transcribable DNA sequence or suppression elementmay comprise a sense sequence that comprises a segment or portion of atargeted GA oxidase gene and an anti-sense sequence that iscomplementary to a segment or portion of the targeted GA oxidase gene,wherein the sense and anti-sense DNA sequences are arranged in tandem.The sense and/or anti-sense sequences, respectively, may each be lessthan 100% identical or complementary to a segment or portion of thetargeted GA oxidase gene as described above. The sense and anti-sensesequences may be separated by a spacer sequence, such that the RNAmolecule transcribed from the suppression element forms a stem, loop orstem-loop structure between the sense and anti-sense sequences. Thesuppression element may instead comprise multiple sense and anti-sensesequences that are arranged in tandem, which may also be separated byone or more spacer sequences. Such suppression elements comprisingmultiple sense and anti-sense sequences may be arranged as a series ofsense sequences followed by a series of anti-sense sequences, or as aseries of tandemly arranged sense and anti-sense sequences.Alternatively, one or more sense DNA sequences may be expressedseparately from the one or more anti-sense sequences (i.e., one or moresense DNA sequences may be expressed from a first transcribable DNAsequence, and one or more anti-sense DNA sequences may be expressed froma second transcribable DNA sequence, wherein the first and secondtranscribable DNA sequences are expressed as separate transcripts).

For suppression of GA oxidase gene(s) using a microRNA (miRNA), thetranscribable DNA sequence or suppression element may comprise a DNAsequence derived from a miRNA sequence native to a virus or eukaryote,such as an animal or plant, or modified or derived from such a nativemiRNA sequence. Such native or native-derived miRNA sequences may form afold back structure and serve as a scaffold for the precursor miRNA(pre-miRNA), and may correspond to the stem region of a native miRNAprecursor sequence, such as from a native (or native-derived)primary-miRNA (pri-miRNA) or pre-miRNA sequence. However, in addition tothese native or native-derived miRNA scaffold or preprocessed sequences,engineered or synthetic miRNAs of the present embodiments furthercomprise a sequence corresponding to a segment or portion of thetargeted GA oxidase gene(s). Thus, in addition to the pre-processed orscaffold miRNA sequences, the suppression element may further comprise asense and/or anti-sense sequence that corresponds to a segment orportion of a targeted GA oxidase gene, and/or a sequence that iscomplementary thereto, although one or more sequence mismatches may betolerated.

Engineered miRNAs are useful for targeted gene suppression withincreased specificity. See, e.g., Parizotto et al., Genes Dev.18:2237-2242 (2004), and U.S. Patent Application Publication Nos.2004/0053411, 2004/0268441, 2005/0144669, and 2005/0037988, the contentsand disclosures of which are incorporated herein by reference. miRNAsare non-protein coding RNAs. When a miRNA precursor molecule is cleaved,a mature miRNA is formed that is typically from about 19 to about 25nucleotides in length (commonly from about 20 to about 24 nucleotides inlength in plants), such as 19, 20, 21, 22, 23, 24, or 25 nucleotides inlength, and has a sequence corresponding to the gene targeted forsuppression and/or its complement. The mature miRNA hybridizes to targetmRNA transcripts and guides the binding of a complex of proteins to thetarget transcripts, which may function to inhibit translation and/orresult in degradation of the transcript, thus negatively regulating orsuppressing expression of the targeted gene. miRNA precursors are alsouseful in plants for directing in-phase production of siRNAs,trans-acting siRNAs (ta-siRNAs), in a process that requires aRNA-dependent RNA polymerase to cause suppression of a target gene. See,e.g., Allen et al., Cell 121:207-221 (2005), Vaucheret Science STKE,2005:pe43 (2005), and Yoshikawa et al. Genes Dev., 19:2164-2175 (2005),the contents and disclosures of which are incorporated herein byreference.

Plant miRNAs regulate their target genes by recognizing and binding to acomplementary or near-perfectly complementary sequence (miRNArecognition site) in the target mRNA transcript, followed by cleavage ofthe transcript by RNase III enzymes, such as ARGONAUTE1. In plants,certain mismatches between a given miRNA recognition site and thecorresponding mature miRNA are typically not tolerated, particularlymismatched nucleotides at positions 10 and 11 of the mature miRNA.Positions within the mature miRNA are given in the 5′ to 3′ direction.Perfect complementarity between a given miRNA recognition site and thecorresponding mature miRNA is usually required at positions 10 and 11 ofthe mature miRNA. See, for example, Franco-Zorrilla et al. (2007) NatureGenetics, 39:1033-1037; and Axtell et al. (2006) Cell, 127:565-577.

Many microRNA genes (MIR genes) have been identified and made publiclyavailable in a database (“miRBase”, available on line atmicrorna.sanger.ac.uk/sequences; also see Griffiths-Jones et al. (2003)Nucleic Acids Res., 31:439-441). MIR genes have been reported to occurin intergenic regions, both isolated and in clusters in the genome, butcan also be located entirely or partially within introns of other genes(both protein-coding and non-protein-coding). For a review of miRNAbiogenesis, see Kim (2005) Nature Rev. Mol. Cell. Biol., 6:376-385.Transcription of MIR genes can be, at least in some cases, underpromotional control of a MIR gene's own promoter. The primarytranscript, termed a “pri-miRNA”, can be quite large (several kilobases)and can be polycistronic, containing one or more pre-miRNAs (fold-backstructures containing a stem-loop arrangement that is processed to themature miRNA) as well as the usual 5′ “cap” and polyadenylated tail ofan mRNA. See, for example, FIG. 1 in Kim (2005) Nature Rev. Mol. Cell.Biol., 6:376-385.

Transgenic expression of miRNAs (whether a naturally occurring sequenceor an artificial sequence) can be employed to regulate expression of themiRNA's target gene or genes. Recognition sites of miRNAs have beenvalidated in all regions of a mRNA, including the 5′ untranslatedregion, coding region, intron region, and 3′ untranslated region,indicating that the position of the miRNA target or recognition siterelative to the coding sequence may not necessarily affect suppression(see, e.g., Jones-Rhoades and Bartel (2004). Mol. Cell, 14:787-799,Rhoades et al. (2002) Cell, 110:513-520, Allen et al. (2004) Nat.Genet., 36:1282-1290, Sunkar and Zhu (2004) Plant Cell, 16:2001-2019).miRNAs are important regulatory elements in eukaryotes, and transgenicsuppression with miRNAs is a useful tool for manipulating biologicalpathways and responses. A description of native miRNAs, theirprecursors, recognition sites, and promoters is provided in U.S. PatentApplication Publication No. 2006/0200878, the contents and disclosuresof which are incorporated herein by reference.

Designing an artificial miRNA sequence can be achieved by substitutingnucleotides in the stem region of a miRNA precursor with a sequence thatis complementary to the intended target, as demonstrated, for example,by Zeng et al. (2002) Mol. Cell, 9:1327-1333. According to manyembodiments, the target may be a sequence of a GA20 oxidase gene or aGA3 oxidase gene. One non-limiting example of a general method fordetermining nucleotide changes in a native miRNA sequence to produce anengineered miRNA precursor for a target of interest includes thefollowing steps: (a) Selecting a unique target sequence of at least 18nucleotides specific to the target gene, e.g., by using sequencealignment tools such as BLAST (see, for example, Altschul et al. (1990)J. Mol. Biol., 215:403-410; Altschul et al. (1997) Nucleic Acids Res.,25:3389-3402); cDNA and/or genomic DNA sequences may be used to identifytarget transcript orthologues and any potential matches to unrelatedgenes, thereby avoiding unintentional silencing or suppression ofnon-target sequences; (b) Analyzing the target gene for undesirablesequences (e.g., matches to sequences from non-target species), andscore each potential target sequence for GC content, Reynolds score (seeReynolds et al. (2004) Nature Biotechnol., 22:326-330), and functionalasymmetry characterized by a negative difference in free energy (“ΔΔG”)(see Khvorova et al. (2003) Cell, 115:209-216). Preferably, targetsequences (e.g., 19-mers) may be selected that have all or most of thefollowing characteristics: (1) a Reynolds score >4, (2) a GC contentbetween about 40% to about 60%, (3) a negative ΔΔG, (4) a terminaladenosine, (5) lack of a consecutive run of 4 or more of the samenucleotide; (6) a location near the 3′ terminus of the target gene; (7)minimal differences from the miRNA precursor transcript. In one aspect,a non-coding RNA molecule used herein to suppress a target gene (e.g., aGA20 or GA3 oxidase gene) is designed to have a target sequenceexhibiting one or more, two or more, three or more, four or more, orfive or more of the foregoing characteristics. Positions at every thirdnucleotide of a suppression element may be important in influencing RNAiefficacy; for example, an algorithm, “siExplorer” is publicly availableat rna.chem.t.u-tokyo.ac.jp/siexplorer.htm (see Katoh and Suzuki (2007)Nucleic Acids Res., 10.1093/nar/gkl1120); (c) Determining a reversecomplement of the selected target sequence (e.g., 19-mer) to use inmaking a modified mature miRNA. Relative to a 19-mer sequence, anadditional nucleotide at position 20 may be matched to the selectedtarget or recognition sequence, and the nucleotide at position 21 may bechosen to either be unpaired to prevent spreading of silencing on thetarget transcript or paired to the target sequence to promote spreadingof silencing on the target transcript; and (d) Transforming theartificial miRNA into a plant.

According to embodiments of the present disclosure, a recombinant DNAmolecule, construct or vector is provided comprising a transcribable DNAsequence or suppression element encoding a miRNA or precursor miRNAmolecule for targeted suppression of a GA oxidase gene(s). Such atranscribable DNA sequence and suppression element may comprise asequence of at least 19 nucleotides in length that corresponds to one ormore GA oxidase gene(s) and/or a sequence complementary to one or moreGA oxidase gene(s), although one or more sequence mismatches ornon-base-paired nucleotides may be tolerated.

GA oxidase gene(s) may also be suppressed using one or more smallinterfering RNAs (siRNAs). The siRNA pathway involves the non-phasedcleavage of a longer double-stranded RNA intermediate (“RNA duplex”)into small interfering RNAs (siRNAs). The size or length of siRNAsranges from about 19 to about 25 nucleotides or base pairs, but commonclasses of siRNAs include those containing 21 or 24 base pairs. Thus, atranscribable DNA sequence or suppression element may encode a RNAmolecule that is at least about 19 to about 25 nucleotides (or more) inlength, such as at least 19, 20, 21, 22, 23, 24, or 25 nucleotides inlength. For siRNA suppression, a recombinant DNA molecule, construct orvector is thus provided comprising a transcribable DNA sequence andsuppression element encoding a siRNA molecule for targeted suppressionof a GA oxidase gene(s). Such a transcribable DNA sequence andsuppression element may be at least 19 nucleotides in length and have asequence corresponding to one or more GA oxidase gene(s), and/or asequence complementary to one or more GA oxidase gene(s).

GA oxidase gene(s) may also be suppressed using one or more trans-actingsmall interfering RNAs (ta-siRNAs). In the ta-siRNA pathway, miRNAsserve to guide in-phase processing of siRNA primary transcripts in aprocess that requires an RNA-dependent RNA polymerase for production ofa double-stranded RNA precursor. ta-siRNAs are defined by lack ofsecondary structure, a miRNA target site that initiates production ofdouble-stranded RNA, requirements of DCL4 and an RNA-dependent RNApolymerase (RDR6), and production of multiple perfectly phased ˜21-ntsmall RNAs with perfectly matched duplexes with 2-nucleotide 3′overhangs (see Allen et al. (2005) Cell, 121:207-221). The size orlength of ta-siRNAs ranges from about 20 to about 22 nucleotides or basepairs, but are mostly commonly 21 base pairs. Thus, a transcribable DNAsequence or suppression element of the present invention may encode aRNA molecule that is at least about 20 to about 22 nucleotides inlength, such as 20, 21, or 22 nucleotides in length. For ta-siRNAsuppression, a recombinant DNA molecule, construct or vector is thusprovided comprising a transcribable DNA sequence or suppression elementencoding a ta-siRNA molecule for targeted suppression of a GA oxidasegene(s). Such a transcribable DNA sequence and suppression element maybe at least 20 nucleotides in length and have a sequence correspondingto one or more GA oxidase gene(s) and/or a sequence complementary to oneor more GA oxidase gene(s). For methods of constructing suitableta-siRNA scaffolds, see, e.g., U.S. Pat. No. 9,309,512, which isincorporated herein by reference in its entirety.

According to embodiments of the present invention, a recombinant DNAmolecule, vector or construct is provided comprising a transcribable DNAsequence encoding a non-coding RNA molecule that binds or hybridizes toa target mRNA in a plant cell, wherein the target mRNA molecule encodesa GA20 or GA3 oxidase gene, and wherein the transcribable DNA sequenceis operably linked to a constitutive or tissue-specific ortissue-preferred promoter. In addition to targeting a mature mRNAsequence, a non-coding RNA molecule may instead target an intronicsequence of a GA oxidase gene or mRNA transcript, or a GA oxidase mRNAsequence overlapping coding and non-coding sequences. According to otherembodiments, a recombinant DNA molecule, vector or construct is providedcomprising a transcribable DNA sequence encoding a non-coding RNA(precursor) molecule that is cleaved or processed into a maturenon-coding RNA molecule that binds or hybridizes to a target mRNA in aplant cell, wherein the target mRNA molecule encodes a GA20 or GA3oxidase protein, and wherein the transcribable DNA sequence is operablylinked to a constitutive or tissue-specific or tissue-preferredpromoter. For purposes of the present disclosure, a “non-coding RNAmolecule” is a RNA molecule that does not encode a protein. Non-limitingexamples of a non-coding RNA molecule include a microRNA (miRNA), amiRNA precursor, a small interfering RNA (siRNA), a siRNA precursor, asmall RNA (18-26 nt in length) and precursors encoding the same, aheterochromatic siRNA (hc-siRNA), a Piwi-interacting RNA (piRNA), ahairpin double strand RNA (hairpin dsRNA), a trans-acting siRNA(ta-siRNA), a naturally occurring antisense siRNA (nat-siRNA), a CRISPRRNA (crRNA), a tracer RNA (tracrRNA), a guide RNA (gRNA), and asingle-guide RNA (sgRNA).

According to embodiments of the present disclosure, suitabletissue-specific or tissue preferred promoters for expression of a GA20oxidase or GA3 oxidase suppression element may include those promotersthat drive or cause expression of its associated suppression element orsequence at least in the vascular and/or leaf tissue(s) of a corn orcereal plant, or possibly other tissues in the case of GA3 oxidase.Expression of the GA oxidase suppression element or construct with atissue-specific or tissue-preferred promoter may also occur in othertissues of the cereal or corn plant outside of the vascular and leaftissues, but active GA levels in the developing reproductive tissues ofthe plant (particularly in the female reproductive organ or ear) arepreferably not significantly reduced or impacted (relative to wild typeor control plants), such that development of the female organ or ear mayproceed normally in the transgenic plant without off-types in the earand a loss in yield potential.

Any vascular promoters known in the art may potentially be used as thetissue-specific or tissue-preferred promoter. Examples of vascularpromoters include the RTBV promoter (see, e.g., SEQ ID NO: 65), a knownsucrose synthase gene promoter, such as a corn sucrose synthase-1 (Sus1or Sh1) promoter (see, e.g., SEQ ID NO: 67), a corn Sh1 gene paralogpromoter, a barley sucrose synthase promoter (Ss1) promoter, a ricesucrose synthase-1 (RSs1) promoter (see, e.g., SEQ ID NO: 68), or a ricesucrose synthase-2 (RSs2) promoter (see, e.g., SEQ ID NO: 69), a knownsucrose transporter gene promoter, such as a rice sucrose transporterpromoter (SUT1) (see, e.g., SEQ ID NO: 70), or various known viralpromoters, such as a Commelina yellow mottle virus (CoYMV) promoter, awheat dwarf geminivirus (WDV) large intergenic region (LIR) promoter, amaize streak geminivirus (MSV) coat protein (CP) promoter, or a riceyellow stripe 1 (YS1)-like or OsYSL2 promoter (SEQ ID NO: 71), and anyfunctional sequence portion or truncation of any of the foregoingpromoters with a similar pattern of expression, such as a truncated RTBVpromoter (see, e.g., SEQ ID NO: 66).

Any leaf promoters known in the art may potentially be used as thetissue-specific or tissue-preferred promoter. Examples of leaf promotersinclude a corn pyruvate phosphate dikinase or PPDK promoter (see, e.g.,SEQ ID NO: 72), a corn fructose 1,6 bisphosphate aldolase or FDApromoter (see, e.g., SEQ ID NO: 73), and a rice Nadh-Gogat promoter(see, e.g., SEQ ID NO: 74), and any functional sequence portion ortruncation of any of the foregoing promoters with a similar pattern ofexpression. Other examples of leaf promoters from monocot plant genesinclude a ribulose biphosphate carboxylase (RuBisCO) or RuBisCO smallsubunit (RBCS) promoter, a chlorophyll a/b binding protein genepromoter, a phosphoenolpyruvate carboxylase (PEPC) promoter, and a Mybgene promoter, and any functional sequence portion or truncation of anyof these promoters with a similar pattern of expression.

Any other vascular and/or leaf promoters known in the art may also beused, including promoter sequences from related genes (e.g., sucrosesynthase, sucrose transporter, and viral gene promoter sequences) fromthe same or different plant species or virus that have a similar patternof expression. Further provided are promoter sequences with a highdegree of homology to any of the foregoing. For example, a vascularpromoter may comprise a DNA sequence that is at least at least 70%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, at least 99.5% or 100% identicalto one or more of SEQ ID NOs: 65, 66, 67, 68, 69, 70, and 71, anyfunctional sequence portion or truncation thereof, and/or any sequencecomplementary to any of the foregoing sequences; a leaf promoter maycomprise, for example, a DNA sequence that is at least at least 70%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, at least 99.5% or 100% identicalto one or more of SEQ ID NOs: 72, 73, and 74, any functional sequenceportion or truncation thereof, and/or any sequence complementary to anyof the foregoing sequences; and a constitutive promoter may comprise aDNA sequence that is at least at least 70%, at least 80%, at least 85%,at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, at least 99.5% or 100% identical to one or more of SEQ IDNOs: 75, 76, 77, 78, 79, 80, 81, 82, and 83, any functional sequenceportion or truncation thereof, and/or any sequence complementary to anyof the foregoing sequences. Examples of vascular and/or leaf promotersmay further include other known, engineered and/or later-identifiedpromoter sequences shown to have a pattern of expression in vascularand/or leaf tissue(s) of a cereal or corn plant. Furthermore, any knownor later-identified constitutive promoter may also be used forexpression of a GA20 oxidase or GA3 oxidase suppression element. Commonexamples of constitutive promoters are provided below.

As understood in the art, the term “promoter” may generally refer to aDNA sequence that contains an RNA polymerase binding site, transcriptionstart site, and/or TATA box and assists or promotes the transcriptionand expression of an associated transcribable polynucleotide sequenceand/or gene (or transgene). A promoter may be synthetic or artificialand/or engineered, varied or derived from a known or naturally occurringpromoter sequence. A promoter may be a chimeric promoter comprising acombination of two or more heterologous sequences. A promoter of thepresent invention may thus include variants of promoter sequences thatare similar in composition, but not identical to, other promotersequence(s) known or provided herein. A promoter may be classifiedaccording to a variety of criteria relating to the pattern of expressionof an associated coding or transcribable sequence or gene (including atransgene) operably linked to the promoter, such as constitutive,developmental, tissue-specific, inducible, etc. Promoters that driveexpression in all or nearly all tissues of the plant are referred to as“constitutive” promoters. However, the expression level with a“constitutive promoter” is not necessarily uniform across differenttissue types and cells. Promoters that drive expression during certainperiods or stages of development are referred to as “developmental”promoters. Promoters that drive enhanced expression in certain tissuesof the plant relative to other plant tissues are referred to as“tissue-enhanced” or “tissue-preferred” promoters. Thus, a“tissue-preferred” promoter causes relatively higher or preferential orpredominant expression in a specific tissue(s) of the plant, but withlower levels of expression in other tissue(s) of the plant. Promotersthat express within a specific tissue(s) of the plant, with little or noexpression in other plant tissues, are referred to as “tissue-specific”promoters. A tissue-specific or tissue-preferred promoter may also bedefined in terms of the specific or preferred tissue(s) in which itdrives expression of its associated transcribable DNA sequence orsuppression element. For example, a promoter that causes specificexpression in vascular tissues may be referred to as a“vascular-specific promoter”, whereas a promoter that causespreferential or predominant expression in vascular tissues may bereferred to as a “vascular-preferred promoter”. Likewise, a promoterthat causes specific expression in leaf tissues may be referred to as a“leaf-specific promoter”, whereas a promoter that causes preferential orpredominant expression in leaf tissues may be referred to as a“leaf-preferred promoter”. An “inducible” promoter is a promoter thatinitiates transcription in response to an environmental stimulus such ascold, drought or light, or other stimuli, such as wounding or chemicalapplication. A promoter may also be classified in terms of its origin,such as being heterologous, homologous, chimeric, synthetic, etc. A“heterologous” promoter is a promoter sequence having a different originrelative to its associated transcribable sequence, coding sequence, orgene (or transgene), and/or not naturally occurring in the plant speciesto be transformed, as defined above.

Several of the GA oxidases in cereal plants consist of a family ofrelated GA oxidase genes. For example, corn has a family of at leastnine GA20 oxidase genes that includes GA20 oxidase_1, GA20 oxidase_2,GA20 oxidase_3, GA20 oxidase_4, GA20 oxidase_5, GA20 oxidase_6, GA20oxidase_7, GA20 oxidase_8, and GA20 oxidase_9. However, there are onlytwo GA3 oxidases in corn, GA3 oxidase_1 and GA3 oxidase_2. The DNA andprotein sequences by SEQ ID NOs for each of these GA20 oxidase genes areprovided in Table 1, and the DNA and protein sequences by SEQ ID NOs foreach of these GA3 oxidase genes are provided in Table 2.

TABLE 1 DNA and protein sequences by sequence identifier for GA20oxidase genes in corn. GA20 oxidase Coding Sequence Gene cDNA (CDS)Protein GA20 oxidase_1 SEQ ID NO: 1 SEQ ID NO: 2 SEQ ID NO: 3 GA20oxidase_2 SEQ ID NO: 4 SEQ ID NO: 5 SEQ ID NO: 6 GA20 oxidase_3 SEQ IDNO: 7 SEQ ID NO: 8 SEQ ID NO: 9 GA20 oxidase_4 SEQ ID NO: 10 SEQ ID NO:11 SEQ ID NO: 12 GA20 oxidase_5 SEQ ID NO: 13 SEQ ID NO: 14 SEQ ID NO:15 GA20 oxidase_6 SEQ ID NO: 16 SEQ ID NO: 17 SEQ ID NO: 18 GA20oxidase_7 SEQ ID NO: 19 SEQ ID NO: 20 SEQ ID NO: 21 GA20 oxidase_8 SEQID NO: 22 SEQ ID NO: 23 SEQ ID NO: 24 GA20 oxidase_9 SEQ ID NO: 25 SEQID NO: 26 SEQ ID NO: 27

TABLE 2 DNA and protein sequences by sequence identifier for GA3 oxidasegenes in corn. GA3 oxidase Coding Sequence Gene cDNA (CDS) Protein GA3oxidase_1 SEQ ID NO: 28 SEQ ID NO: 29 SEQ ID NO: 30 GA3 oxidase_2 SEQ IDNO: 31 SEQ ID NO: 32 SEQ ID NO: 33

The genomic DNA sequence of GA20 oxidase_3 is provided in SEQ ID NO: 34,and the genomic DNA sequence of GA20 oxidase_5 is provided in SEQ ID NO:35. For the GA20 oxidase_3 gene, SEQ ID NO: 34 provides 3000 nucleotidesupstream of the GA20 oxidase_3 5′-UTR; nucleotides 3001-3096 correspondto the 5′-UTR; nucleotides 3097-3665 correspond to the first exon;nucleotides 3666-3775 correspond to the first intron; nucleotides3776-4097 correspond to the second exon; nucleotides 4098-5314correspond to the second intron; nucleotides 5315-5584 correspond to thethird exon; and nucleotides 5585-5800 correspond to the 3′-UTR. SEQ IDNO: 34 also provides 3000 nucleotides downstream of the end of the3′-UTR (nucleotides 5801-8800). For the GA20 oxidase_5 gene, SEQ ID NO:35 provides 3000 nucleotides upstream of the GA20 oxidase_5 start codon(nucleotides 1-3000); nucleotides 3001-3791 correspond to the firstexon; nucleotides 3792-3906 correspond to the first intron; nucleotides3907-4475 correspond to the second exon; nucleotides 4476-5197correspond to the second intron; nucleotides 5198-5473 correspond to thethird exon; and nucleotides 5474-5859 correspond to the 3′-UTR. SEQ IDNO: 35 also provides 3000 nucleotides downstream of the end of the3′-UTR (nucleotides 5860-8859).

The genomic DNA sequence of GA3 oxidase_1 is provided in SEQ ID NO: 36,and the genomic DNA sequence of GA3 oxidase_2 is provided in SEQ ID NO:37. For the GA3 oxidase_1 gene, nucleotides 1-29 of SEQ ID NO: 36correspond to the 5′-UTR; nucleotides 30-514 of SEQ ID NO: 36 correspondto the first exon; nucleotides 515-879 of SEQ ID NO: 36 correspond tothe first intron; nucleotides 880-1038 of SEQ ID NO: 36 correspond tothe second exon; nucleotides 1039-1158 of SEQ ID NO: 36 correspond tothe second intron; nucleotides 1159-1663 of SEQ ID NO: 36 correspond tothe third exon; and nucleotides 1664-1788 of SEQ ID NO: 36 correspond tothe 3′-UTR. For the GA3 oxidase_2 gene, nucleotides 1-38 of SEQ ID NO:37 correspond to the 5-UTR; nucleotides 39-532 of SEQ ID NO: 37correspond to the first exon; nucleotides 533-692 of SEQ ID NO: 37correspond to the first intron; nucleotides 693-851 of SEQ ID NO: 37correspond to the second exon; nucleotides 852-982 of SEQ ID NO: 37correspond to the second intron; nucleotides 983-1445 of SEQ ID NO: 37correspond to the third exon; and nucleotides 1446-1698 of SEQ ID NO: 37correspond to the 3′-UTR.

In addition to phenotypic observations with targeting the GA20 oxidase_3and/or GA20 oxidase_5 gene(s), or the GA3 oxidase_1 and/or GA3 oxidase_2gene(s), for suppression, a semi-dwarf phenotype is also observed withsuppression of the GA20 oxidase_4 gene. The genomic DNA sequence of GA20oxidase_4 is provided in SEQ ID NO: 38. For the GA oxidase_4 gene, SEQID NO: 38 provides nucleotides 1-1416 upstream of the 5′-UTR;nucleotides 1417-1543 of SEQ ID NO: 38 correspond to the 5′-UTR;nucleotides 1544-1995 of SEQ ID NO: 38 correspond to the first exon;nucleotides 1996-2083 of SEQ ID NO: 38 correspond to the first intron;nucleotides 2084-2411 of SEQ ID NO: 38 correspond to the second exon;nucleotides 2412-2516 of SEQ ID NO: 38 correspond to the second intron;nucleotides 2517-2852 of SEQ ID NO: 38 correspond to the third exon;nucleotides 2853-3066 of SEQ ID NO: 38 correspond to the 3′-UTR; andnucleotides 3067-4465 of SEQ ID NO: 38 corresponds to genomic sequencedownstream of to the 3′-UTR.

According to embodiments of the present disclosure, a recombinant DNAmolecule, vector or construct is provided comprising a transcribable DNAsequence encoding a non-coding RNA molecule, wherein the non-coding RNAmolecule comprises a sequence that is at least 80%, at least 85%, atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, at least 99.5%, or 100% complementary to at least a segmentor portion of a mRNA molecule (i) expressed from an endogenous GAoxidase gene and/or (ii) encoding an endogenous GA oxidase protein inthe plant, wherein the transcribable DNA sequence is operably linked toa plant-expressible promoter, and wherein the plant is a cereal or cornplant.

According to some embodiments, a non-coding RNA molecule targets GA20oxidase gene(s), such as GA20 oxidase_3 and/or GA20 oxidase_5 gene(s),for suppression and comprises a sequence that is at least 80%, at least85%, at least 90%, at least 95%, at least 96%, at least 97%, at least98%, at least 99%, at least 99.5%, or 100% complementary to at least 15,at least 16, at least 17, at least 18, at least 19, at least 20, atleast 21, at least 22, at least 23, at least 24, at least 25, at least26, or at least 27 consecutive nucleotides of one or more of SEQ ID NOs:7, 8, 13 and 14. According to some embodiments, a non-coding RNAmolecule is at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or100% complementary to at least 15, at least 16, at least 17, at least18, at least 19, at least 20, at least 21, at least 22, at least 23, atleast 24, at least 25, at least 26, or at least 27 consecutivenucleotides of a mRNA molecule encoding an endogenous GA20 oxidaseprotein in the plant that is at least 80%, at least 85%, at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, atleast 99.5%, or 100% identical to one or both of SEQ ID NOs: 9 and 15.According to further embodiments, a non-coding RNA molecule maycomprises a sequence that is at least 80%, at least 85%, at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, atleast 99.5%, or 100% complementary to at least 15, at least 16, at least17, at least 18, at least 19, at least 20, at least 21, at least 22, atleast 23, at least 24, at least 25, at least 26, or at least 27consecutive nucleotides of a mRNA molecule encoding an endogenous GA20oxidase protein in the plant that is at least 80%, at least 85%, atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, at least 99.5%, or 100% similar to one or both of SEQ ID NOs:9 and 15. In addition to targeting a mature mRNA sequence (includingeither or both of the untranslated or exonic sequences), a non-codingRNA molecule may further target the intronic sequences of a GA20 oxidasegene or transcript.

According to some embodiments, a non-coding RNA molecule targets GA3oxidase gene(s) for suppression and comprises a sequence that is atleast 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, at least 99.5%, or 100%complementary to at least 15, at least 16, at least 17, at least 18, atleast 19, at least 20, at least 21, at least 22, at least 23, at least24, at least 25, at least 26, or at least 27 consecutive nucleotides ofone or more of SEQ ID NOs: 28, 29, 31 and 32. According to otherembodiments, a non-coding RNA molecule is at least 80%, at least 85%, atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, at least 99.5%, or 100% complementary to at least 15, atleast 16, at least 17, at least 18, at least 19, at least 20, at least21, at least 22, at least 23, at least 24, at least 25, at least 26, orat least 27 consecutive nucleotides of a mRNA molecule encoding anendogenous GA3 oxidase protein in the plant that is at least 80%, atleast 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, at least 99.5%, or 100% identical to one orboth of SEQ ID NOs: 30 and 33. According to further embodiments, anon-coding RNA molecule may comprises a sequence that is at least 80%,at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, at least 99.5%, or 100% complementary to atleast 15, at least 16, at least 17, at least 18, at least 19, at least20, at least 21, at least 22, at least 23, at least 24, at least 25, atleast 26, or at least 27 consecutive nucleotides of a mRNA moleculeencoding an endogenous GA3 oxidase protein in the plant that is at least80%, at least 85%, at least 90%, at least 95%, at least 96%, at least97%, at least 98%, at least 99%, at least 99.5%, or 100% similar to oneor both of SEQ ID NOs: 30 and 33. In addition to targeting a mature mRNAsequence (including either or both of the untranslated or exonicsequences), a non-coding RNA molecule may further target the intronicsequences of a GA3 oxidase gene or transcript.

According to some embodiments, a non-coding RNA molecule targets GA20oxidase_4 gene for suppression and comprises a sequence that is at least80%, at least 85%, at least 90%, at least 95%, at least 96%, at least97%, at least 98%, at least 99%, at least 99.5%, or 100% complementaryto at least 15, at least 16, at least 17, at least 18, at least 19, atleast 20, at least 21, at least 22, at least 23, at least 24, at least25, at least 26, or at least 27 consecutive nucleotides of one or bothof SEQ ID NOs: 10 and 11. According to other embodiments, a non-codingRNA molecule is at least 80%, at least 85%, at least 90%, at least 95%,at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%,or 100% complementary to at least 15, at least 16, at least 17, at least18, at least 19, at least 20, at least 21, at least 22, at least 23, atleast 24, at least 25, at least 26, or at least 27 consecutivenucleotides of a mRNA molecule encoding an endogenous GA20 oxidaseprotein in the plant that is at least 80%, at least 85%, at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, atleast 99.5%, or 100% identical to one or both of SEQ ID NO: 12.According to further embodiments, a non-coding RNA molecule maycomprises a sequence that is at least 80%, at least 85%, at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, atleast 99.5%, or 100% complementary to at least 15, at least 16, at least17, at least 18, at least 19, at least 20, at least 21, at least 22, atleast 23, at least 24, at least 25, at least 26, or at least 27consecutive nucleotides of a mRNA molecule encoding an endogenous GA20oxidase protein in the plant that is at least 80%, at least 85%, atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, at least 99.5%, or 100% similar to SEQ ID NOs: 12. Inaddition to targeting a mature mRNA sequence (including either or bothof the untranslated or exonic sequences), a non-coding RNA molecule mayfurther target the intronic sequences of a GA20 oxidase gene ortranscript.

According to many embodiments, the non-coding RNA molecule encoded bythe transcribable DNA sequence of the recombinant DNA molecule, vectoror construct may be a precursor miRNA or siRNA that is processed orcleaved in a plant cell to form a mature miRNA or siRNA that targets aGA20 oxidase or GA3 oxidase gene.

According to embodiments of the present invention, GA levels may bereduced in the stalk or stem of a cereal or corn plant by targeting onlya limited subset of genes within a GA oxidase family for suppression.Without being bound by theory, it is proposed that targeting of alimited number of genes within a GA oxidase family for suppression mayproduce the short stature phenotype and resistance to lodging intransgenic plants, but without off-types in the reproductive or eartissues of the plant due to differential expression among GA oxidasegenes, sufficient compensation for the suppressed GA oxidase gene(s) byother GA oxidase gene(s) in those reproductive tissues, and/orincomplete suppression of the targeted GA oxidase gene(s). Thus, notonly may off-types be avoided by limiting expression or suppression ofGA oxidase gene(s) with a tissue-specific or tissue preferred promoter,it is proposed that a limited subset of GA oxidase genes (e.g., alimited number of GA20 oxidase genes) may be targeted for suppression,such that the other GA oxidase genes within the same gene family (e.g.,other GA20 oxidase genes) may compensate for loss of expression of thesuppressed GA oxidase gene(s) in those tissues. Incomplete suppressionof the targeted GA oxidase gene(s) may also allow for a sufficient levelof expression of the targeted GA oxidase gene(s) in one or more tissuesto avoid off-types or undesirable traits in the plant that wouldnegatively affect crop yield, such as reproductive off-types orexcessive shortening of plant height. Unlike complete loss-of-functionmutations in a gene, suppression may allow for partial activity of thetargeted gene to persist. Since the different GA20 oxidase genes havedifferent patterns of expression in plants, targeting of a limitedsubset of GA20 oxidase genes for suppression may allow for modificationof certain traits while avoiding off-types previously associated with GAmutants in cereal plants. In other words, the growth, developmental andreproductive traits or off-types previously associated with GA mutantsin corn and other cereal crops may be decoupled by targeting only alimited number or subset (i.e., one or more, but not all) of the GA20 orGA3 oxidase genes and/or by incomplete suppression of a targeted GAoxidase gene. By transgenically targeting a subset of one or moreendogenous GA3 or GA20 oxidase genes for suppression within a plant, amore pervasive pattern of expression (e.g., with a constitutivepromoter) may be used to produce semi-dwarf plants without significantreproductive off-types and/or other undesirable traits in the plant,even with expression of the transgenic construct in reproductivetissue(s). Indeed, suppression elements and constructs are providedherein that selectively target the GA20 oxidase_3 and/or GA20 oxidase_5genes (identified in Table 1 above) for suppression, which may beoperably linked to a vascular, leaf and/or constitutive promoter.

With a suppression construct that only targets a limited subset of GA20oxidase genes, such as the GA20 oxidase_3, GA20 oxidase_4, and/or GA20oxidase_5 gene(s), or which targets the GA3 oxidase_1 and/or GA3oxidase_2 gene(s), restricting the pattern of expression of thesuppression element may be less crucial for obtaining normalreproductive development of the cereal or corn plant and avoidance ofoff-types in the female organ or ear due to compensation, etc., from theother GA20 and/or GA3 oxidase genes. Therefore, expression of asuppression construct and element, selectively or preferentiallytargeting, for instance, the GA20 oxidase_3 and/or GA20 oxidase_5gene(s), the GA20 oxidase_4 gene, and/or the GA3 oxidase_1 and/or GA3oxidase_2 gene(s) in corn, or similar genes and homologs in other cerealplants, may be driven by a variety of different plant-expressiblepromoter types including constitutive and tissue-specific ortissue-preferred promoters, such as a vascular or leaf promoter, whichmay include, for example, the RTBV promoter introduced above (e.g., apromoter comprising the RTBV (SEQ ID NO: 65) or truncated RTBV (SEQ IDNO: 66) sequence), and any other promoters that drive expression intissues encompassing much or all of the vascular and/or leaf tissue(s)of a plant. Any known or later-identified constitutive promoter with asufficiently high level of expression may also be used for expression ofa suppression construct targeting a subset of GA20 and/or GA3 oxidasegenes in corn, particularly the GA20 oxidase_3 and/or GA20 oxidase_5gene(s), the GA20 oxidase_4 gene, and/or the GA3 oxidase_1 and/or GA3oxidase_2 gene(s), or similar genes and homologs in other cereal plants.

Examples of constitutive promoters that may be used in monocot plants,such as cereal or corn plants, include, for example, various actin genepromoters, such as a rice Actin 1 promoter (see, e.g., U.S. Pat. No.5,641,876; see also SEQ ID NO: 75 or SEQ ID NO: 76) and a rice Actin 2promoter (see, e.g., U.S. Pat. No. 6,429,357; see also, e.g., SEQ ID NO:77 or SEQ ID NO: 78), a CaMV 35S or 19S promoter (see, e.g., U.S. Pat.No. 5,352,605; see also, e.g., SEQ ID NO: 79 for CaMV 35S), a maizeubiquitin promoter (see, e.g., U.S. Pat. No. 5,510,474), a Coix lacryma-jobi polyubiquitin promoter (see, e.g., SEQ ID NO: 80), a rice or maizeGos2 promoter (see, e.g., Pater et al., The Plant Journal, 2(6): 837-441992; see also, e.g., SEQ ID NO: 81 for the rice Gos2 promoter), a FMV35S promoter (see, e.g., U.S. Pat. No. 6,372,211), a dual enhanced CMVpromoter (see, e.g., U.S. Pat. No. 5,322,938), a MMV promoter (see,e.g., U.S. Pat. No. 6,420,547; see also, e.g., SEQ ID NO: 82), a PCLSVpromoter (see, e.g., U.S. Pat. No. 5,850,019; see also, e.g., SEQ ID NO:83), an Emu promoter (see, e.g., Last et al., Theor. Appl. Genet. 81:581(1991); and Mcelroy et al., Mol. Gen. Genet. 231:150 (1991)), a tubulinpromoter from maize, rice or other species, a nopaline synthase (nos)promoter, an octopine synthase (ocs) promoter, a mannopine synthase(mas) promoter, or a plant alcohol dehydrogenase (e.g., maize Adh1)promoter, any other promoters including viral promoters known orlater-identified in the art to provide constitutive expression in acereal or corn plant, any other constitutive promoters known in the artthat may be used in monocot or cereal plants, and any functionalsequence portion or truncation of any of the foregoing promoters.

A sufficient level of expression of a transcribable DNA sequenceencoding a non-coding RNA molecule targeting a GA oxidase gene forsuppression may be necessary to produce a short stature, semi-dwarfphenotype that resists lodging, since lower levels of expression may beinsufficient to lower active GA levels in the plant to a sufficientextent to cause a significant phenotype. Thus, tissue-specific andtissue-preferred promoters that drive, etc., a moderate or strong levelof expression of their associated transcribable DNA sequence in activeGA-producing tissue(s) of a plant may be preferred. Furthermore, suchtissue-specific and tissue-preferred should drive, etc., expression oftheir associated transcribable DNA sequence during one or morevegetative stage(s) of plant development when the plant is growingand/or elongating including one or more of the following vegetativestage(s): V_(E), V1, V2, V3, V4, V5, V6, V7, V8, V9, V10, V11, V12, V13,V14, Vn, V_(T), such as expression at least during V3-V12, V4-V12,V5-V12, V6-V12, V7-V12, V8-V12, V3-V14, V5-V14, V6-V14, V7-V14, V8-V14,V9-V14, V10-V14, etc., or during any other range of vegetative stageswhen growth and/or elongation of the plant is occurring.

According to many embodiments, the plant-expressible promoter maypreferably drive expression constitutively or in at least a portion ofthe vascular and/or leaf tissues of the plant. Different promotersdriving expression of a suppression element targeting the endogenousGA20 oxidase_3 and/or GA20 oxidase_5 gene(s), the GA20 oxidase_4 gene,the GA3 oxidase_1 and/or GA3 oxidase_2 gene(s) in corn, or similar genesand homologs in other cereal plants, may be effective at reducing plantheight and increasing lodging resistance to varying degrees depending ontheir particular pattern and strength of expression in the plant.However, some tissue-specific and tissue-preferred promoters drivingexpression of a GA20 or GA3 oxidase suppression element in a plant maynot produce a significant short stature or anti-lodging phenotypes dueto the spatial-temporal pattern of expression of the promoter duringplant development, and/or the amount or strength of expression of thepromoter being too low or weak. Furthermore, some suppression constructsmay only reduce and not eliminate expression of the targeted GA20 or GA3oxidase gene(s) when expressed in a plant, and thus depending on thepattern and strength of expression with a given promoter, the patternand level of expression of the GA20 or GA3 oxidase suppression constructwith such a promoter may not be sufficient to produce an observableplant height and lodging resistance phenotype in plants.

According to present embodiments, a recombinant DNA molecule, vector orconstruct for suppression of one or more endogenous GA20 or GA3 oxidasegene(s) in a plant is provided comprising a transcribable DNA sequenceencoding a non-coding RNA molecule, wherein the non-coding RNA moleculecomprises a sequence that is at least 80%, at least 85%, at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, atleast 99.5%, or 100% complementary to at least a segment or portion of amRNA molecule expressed from an endogenous GA oxidase gene and encodingan endogenous GA oxidase protein in the plant, wherein the transcribableDNA sequence is operably linked to a plant-expressible promoter, andwherein the plant is a cereal or corn plant. As stated above, inaddition to targeting a mature mRNA sequence, a non-coding RNA moleculemay further target the intronic sequence(s) of a GA oxidase gene ortranscript. According to many embodiments, a non-coding RNA molecule maytarget a GA20 oxidase_3 gene for suppression and comprise a sequencethat is at least 80%, at least 85%, at least 90%, at least 95%, at least96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100%complementary to at least 15, at least 16, at least 17, at least 18, atleast 19, at least 20, at least 21, at least 22, at least 23, at least24, at least 25, at least 26, or at least 27 consecutive nucleotides ofSEQ ID NO: 7 or SEQ ID NO: 8. According to some embodiments, anon-coding RNA molecule targeting a GA20 oxidase_3 gene for suppressionmay be complementary to at least 19 consecutive nucleotides, but no morethan 27 consecutive nucleotides, such as complementary to 19, 20, 21,22, 23, 24, 25, 26, or 27 consecutive nucleotides, of SEQ ID NO: 7 orSEQ ID NO: 8. According to some embodiments, a non-coding RNA moleculemay target a GA20 oxidase gene for suppression and comprise a sequencethat is at least 80%, at least 85%, at least 90%, at least 95%, at least96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100%complementary to at least 15, at least 16, at least 17, at least 18, atleast 19, at least 20, at least 21, at least 22, at least 23, at least24, at least 25, at least 26, or at least 27 consecutive nucleotides ofa mRNA molecule encoding an endogenous GA20 oxidase protein in the plantthat is at least 80%, at least 85%, at least 90%, at least 95%, at least96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100%identical to SEQ ID NO: 9. According to further embodiments, anon-coding RNA molecule may comprise a sequence that is at least 80%, atleast 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, at least 99.5%, or 100% complementary to atleast 15, at least 16, at least 17, at least 18, at least 19, at least20, at least 21, at least 22, at least 23, at least 24, at least 25, atleast 26, or at least 27 consecutive nucleotides of a mRNA moleculeencoding an endogenous GA20 oxidase protein that is at least 80%, atleast 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, at least 99.5%, or 100% similar to SEQ ID NO:9.

As mentioned above, a non-coding RNA molecule may target an intronsequence of a GA oxidase gene instead of, or in addition to, an exonic,5′ UTR or 3′ UTR of the GA oxidase gene. Thus, a non-coding RNA moleculetargeting the GA20 oxidase_3 gene for suppression may comprise asequence that is at least 80%, at least 85%, at least 90%, at least 95%,at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%,or 100% complementary to at least 15, at least 16, at least 17, at least18, at least 19, at least 20, at least 21, at least 22, at least 23, atleast 24, at least 25, at least 26, or at least 27 consecutivenucleotides of SEQ ID NO: 34, and/or of nucleotides 3666-3775 or4098-5314 of SEQ ID NO: 34. It is important to note that the sequencesprovided herein for the GA20 oxidase_3 gene may vary across thediversity of corn plants, lines and germplasms due to polymorphismsand/or the presence of different alleles of the gene. Furthermore, aGA20 oxidase_3 gene may be expressed as alternatively spliced isoformsthat may give rise to different mRNA, cDNA and coding sequences that canaffect the design of a suppression construct and non-coding RNAmolecule. Thus, a non-coding RNA molecule targeting a GA20 oxidase_3gene for suppression may be more broadly defined as comprising asequence that is at least 80%, at least 85%, at least 90%, at least 95%,at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%,or 100% complementary to at least 15, at least 16, at least 17, at least18, at least 19, at least 20, at least 21, at least 22, at least 23, atleast 24, at least 25, at least 26, or at least 27 consecutivenucleotides of SEQ ID NO: 34.

According to embodiments of the present disclosure, a recombinant DNAmolecule, vector or construct for suppression of an endogenous GA20oxidase_5 gene in a plant is provided comprising a transcribable DNAsequence encoding a non-coding RNA molecule, wherein the non-coding RNAmolecule targeting the GA20 oxidase_5 gene for suppression comprises asequence that is at least 80%, at least 85%, at least 90%, at least 95%,at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%,or 100% complementary to at least 15, at least 16, at least 17, at least18, at least 19, at least 20, at least 21, at least 22, at least 23, atleast 24, at least 25, at least 26, or at least 27 consecutivenucleotides of SEQ ID NO: 13 or SEQ ID NO: 14. According to someembodiments, a non-coding RNA molecule targeting the GA20 oxidase_5 genefor suppression may be complementary to at least 19 consecutivenucleotides, but no more than 27 consecutive nucleotides, such ascomplementary to 19, 20, 21, 22, 23, 24, 25, 26, or 27 consecutivenucleotides, of SEQ ID NO: 13 or SEQ ID NO: 14. According to someembodiments, a non-coding RNA molecule may target a GA20 oxidase genefor suppression comprise a sequence that is at least 80%, at least 85%,at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, at least 99.5%, or 100% complementary to at least 15, atleast 16, at least 17, at least 18, at least 19, at least 20, at least21, at least 22, at least 23, at least 24, at least 25, at least 26, orat least 27 consecutive nucleotides of a mRNA molecule encoding anendogenous GA20 oxidase protein in the plant that is at least 80%, atleast 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO:15. According to further embodiments, a non-coding RNA molecule maycomprise a sequence that is at least 80%, at least 85%, at least 90%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, atleast 99.5%, or 100% complementary to at least 15, at least 16, at least17, at least 18, at least 19, at least 20, at least 21, at least 22, atleast 23, at least 24, at least 25, at least 26, or at least 27consecutive nucleotides of a mRNA molecule encoding an endogenous GA20oxidase protein that is at least 80%, at least 85%, at least 90%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, atleast 99.5%, or 100% similar to SEQ ID NO: 15.

As mentioned above, a non-coding RNA molecule may target an intronsequence of a GA oxidase gene instead of, or in addition to, an exonicor untranslated region of the mature mRNA of the GA oxidase gene. Thus,a non-coding RNA molecule targeting the GA20 oxidase_5 gene forsuppression may comprise a sequence that is at least 80%, at least 85%,at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, at least 99.5%, or 100% complementary to at least 15, atleast 16, at least 17, at least 18, at least 19, at least 20, at least21, at least 22, at least 23, at least 24, at least 25, at least 26, orat least 27 consecutive nucleotides of SEQ ID NO: 35, and/or ofnucleotides 3792-3906 or 4476-5197 of SEQ ID NO: 35. The sequencesprovided herein for GA20 oxidase_5 may vary across the diversity of cornplants, lines and germplasms due to polymorphisms and/or the presence ofdifferent alleles of the gene. Furthermore, a GA20 oxidase_5 gene may beexpressed as alternatively spliced isoforms that may give rise todifferent mRNA, cDNA and coding sequences that can affect the design ofa suppression construct and non-coding RNA molecule. Thus, a non-codingRNA molecule targeting a GA20 oxidase_3 gene for suppression may bedefined more broadly as comprising a sequence that is at least 80%, atleast 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, at least 99.5%, or 100% complementary to atleast 15, at least 16, at least 17, at least 18, at least 19, at least20, at least 21, at least 22, at least 23, at least 24, at least 25, atleast 26, or at least 27 consecutive nucleotides of SEQ ID NO: 35.

According to further embodiments, a recombinant DNA molecule, vector orconstruct for joint suppression of endogenous GA20 oxidase_3 and GA20oxidase_5 genes in a plant is provided comprising a transcribable DNAsequence encoding a non-coding RNA molecule, wherein the non-coding RNAmolecule targeting the GA20 oxidase_3 and GA20 oxidase_5 genes forsuppression comprises a sequence that is (i) at least 80%, at least 85%,at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, at least 99.5%, or 100% complementary to at least 15, atleast 16, at least 17, at least 18, at least 19, at least 20, at least21, at least 22, at least 23, at least 24, at least 25, at least 26, orat least 27 consecutive nucleotides of SEQ ID NO: 7 and/or SEQ ID NO: 8,and (ii) at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or100% complementary to at least 15, at least 16, at least 17, at least18, at least 19, at least 20, at least 21, at least 22, at least 23, atleast 24, at least 25, at least 26, or at least 27 consecutivenucleotides of SEQ ID NO: 13 and/or SEQ ID NO: 14. According to some ofthese embodiments, the non-coding RNA molecule jointly targeting theGA20 oxidase_3 and GA20 oxidase_5 genes for suppression may becomplementary to at least 19 consecutive nucleotides, but no more than27 consecutive nucleotides, such as complementary to 19, 20, 21, 22, 23,24, 25, 26, or 27 consecutive nucleotides, of (i) SEQ ID NO: 7 (and/orSEQ ID NO: 8) and (ii) SEQ ID NO: 13 (and/or SEQ ID NO: 14). Accordingto many embodiments, the non-coding RNA molecule jointly targeting theGA20 oxidase_3 and GA20 oxidase_5 genes for suppression comprises asequence that is (i) at least 80%, at least 85%, at least 90%, at least95%, at least 96%, at least 97%, at least 98%, at least 99%, at least99.5%, or 100% complementary to at least 15, at least 16, at least 17,at least 18, at least 19, at least 20, at least 21, at least 22, atleast 23, at least 24, at least 25, at least 26, or at least 27consecutive nucleotides of a mRNA molecule encoding an endogenous GA20oxidase protein in the plant that is at least 80%, at least 85%, atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, at least 99.5%, or 100% identical to SEQ ID NO: 9, and (ii)at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, at least 99.5%, or 100%complementary to at least 15, at least 16, at least 17, at least 18, atleast 19, at least 20, at least 21, at least 22, at least 23, at least24, at least 25, at least 26, or at least 27 consecutive nucleotides ofa mRNA molecule encoding an endogenous GA20 oxidase protein in the plantthat is at least 80%, at least 85%, at least 90%, at least 95%, at least96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100%identical to SEQ ID NO: 15. As mentioned above, the non-coding RNAmolecule may target an intron sequence of a GA oxidase gene. Thus, thenon-coding RNA molecule may target an intron sequence(s) of one or bothof the GA20 oxidase_3 and/or GA20 oxidase_5 gene(s) as identified above.

According to particular embodiments, the non-coding RNA molecule encodedby a transcribable DNA sequence comprises (i) a sequence that is atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, atleast 99.5%, or 100% complementary to SEQ ID NO: 39, 41, 43 or 45,and/or (ii) a sequence or suppression element encoding a non-coding RNAmolecule comprising a sequence that is at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, at least 99.5%, or 100% identicalto SEQ ID NO: 40, 42, 44 or 46. According to some embodiments, thenon-coding RNA molecule encoded by a transcribable DNA sequence maycomprise a sequence with one or more mismatches, such as 1, 2, 3, 4, 5or more complementary mismatches, relative to the sequence of a targetor recognition site of a targeted GA20 oxidase gene mRNA, such as asequence that is nearly complementary to SEQ ID NO: 40 but with one ormore complementary mismatches relative to SEQ ID NO: 40. According to aparticular embodiment, the non-coding RNA molecule encoded by thetranscribable DNA sequence comprises a sequence that is 100% identicalto SEQ ID NO: 40, which is 100% complementary to a target sequencewithin the cDNA and coding sequences of the GA20 oxidase_3 (i.e., SEQ IDNOs: 7 and 8, respectively), and/or to a corresponding sequence of amRNA encoded by an endogenous GA20 oxidase_3 gene. However, the sequenceof a non-coding RNA molecule encoded by a transcribable DNA sequencethat is 100% identical to SEQ ID NO: 40, 42, 44 or 46 may not beperfectly complementary to a target sequence within the cDNA and codingsequences of the GA20 oxidase_5 gene (i.e., SEQ ID NOs: 13 and 14,respectively), and/or to a corresponding sequence of a mRNA encoded byan endogenous GA20 oxidase_5 gene. For example, the closestcomplementary match between the non-coding RNA molecule or miRNAsequence in SEQ ID NO: 40 and the cDNA and coding sequences of the GA20oxidase_5 gene may include one mismatch at the first position of SEQ IDNO: 39 (i.e., the “C” at the first position of SEQ ID NO: 39 is replacedwith a “G”; i.e., GTCCATCATGCGGTGCAACTA). However, the non-coding RNAmolecule or miRNA sequence in SEQ ID NO: 40 may still bind and hybridizeto the mRNA encoded by the endogenous GA20 oxidase_5 gene despite thisslight mismatch.

According to embodiments of the present disclosure, a recombinant DNAmolecule, vector or construct for suppression of one or more endogenousGA3 oxidase gene(s) in a plant is provided comprising a transcribableDNA sequence encoding a non-coding RNA molecule, wherein the non-codingRNA molecule comprises a sequence that is at least 80%, at least 85%, atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, at least 99.5%, or 100% complementary to at least a segmentor portion of a mRNA molecule expressed from an endogenous GA3 oxidasegene and encoding an endogenous GA3 oxidase protein in the plant,wherein the transcribable DNA sequence is operably linked to aplant-expressible promoter, and wherein the plant is a cereal or cornplant. In addition to targeting a mature mRNA sequence, a non-coding RNAmolecule may further target the intronic sequences of a GA3 oxidase geneor transcript.

According to some embodiments, a non-coding RNA molecule may target aGA3 oxidase_1 gene for suppression and comprise a sequence that is atleast 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, at least 99.5%, or 100%complementary to at least 15, at least 16, at least 17, at least 18, atleast 19, at least 20, at least 21, at least 22, at least 23, at least24, at least 25, at least 26, or at least 27 consecutive nucleotides ofSEQ ID NO: 28 or SEQ ID NO: 29. According to some embodiments, anon-coding RNA molecule targeting a GA3 oxidase gene for suppression maybe complementary to at least 19 consecutive nucleotides, but no morethan 27 consecutive nucleotides, such as complementary to 19, 20, 21,22, 23, 24, 25, 26, or 27 consecutive nucleotides, of SEQ ID NO: 28 orSEQ ID NO: 29. According to some embodiments, a non-coding RNA moleculetargeting a GA3 oxidase gene for suppression comprises a sequence thatis at least 80%, at least 85%, at least 90%, at least 95%, at least 96%,at least 97%, at least 98%, at least 99%, at least 99.5%, or 100%complementary to at least 15, at least 16, at least 17, at least 18, atleast 19, at least 20, at least 21, at least 22, at least 23, at least24, at least 25, at least 26, or at least 27 consecutive nucleotides ofa mRNA molecule encoding an endogenous GA3 oxidase protein in the plantthat is at least 80%, at least 85%, at least 90%, at least 95%, at least96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100%identical to SEQ ID NO: 30. According to further embodiments, anon-coding RNA molecule may comprise a sequence that is at least 80%, atleast 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, at least 99.5%, or 100% complementary to atleast 15, at least 16, at least 17, at least 18, at least 19, at least20, at least 21, at least 22, at least 23, at least 24, at least 25, atleast 26, or at least 27 consecutive nucleotides of a mRNA moleculeencoding an endogenous GA3 oxidase protein that is at least 80%, atleast 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, at least 99.5%, or 100% similar to SEQ ID NO:30.

As mentioned above, a non-coding RNA molecule may target an intronsequence of a GA3 oxidase gene instead of, or in addition to, an exonic,5′ UTR or 3′ UTR of the GA oxidase gene. Thus, a non-coding RNA moleculetargeting the GA3 oxidase_1 gene for suppression may comprise a sequencethat is at least 80%, at least 85%, at least 90%, at least 95%, at least96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100%complementary to at least 15, at least 16, at least 17, at least 18, atleast 19, at least 20, at least 21, at least 22, at least 23, at least24, at least 25, at least 26, or at least 27 consecutive nucleotides ofSEQ ID NO: 36, and/or of nucleotides 515-879 or 1039-1158 of SEQ ID NO:36. The sequences provided herein for GA3 oxidase_1 may vary across thediversity of corn plants, lines and germplasms due to polymorphismsand/or the presence of different alleles of the gene. Furthermore, a GA3oxidase_1 gene may be expressed as alternatively spliced isoforms thatmay give rise to different mRNA, cDNA and coding sequences that canaffect the design of a suppression construct and non-coding RNAmolecule. Thus, a non-coding RNA molecule targeting a GA3 oxidase_1 genefor suppression may be defined more broadly as comprising a sequencethat is at least 80%, at least 85%, at least 90%, at least 95%, at least96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100%complementary to at least 15, at least 16, at least 17, at least 18, atleast 19, at least 20, at least 21, at least 22, at least 23, at least24, at least 25, at least 26, or at least 27 consecutive nucleotides ofSEQ ID NO: 36.

According to some embodiments, a non-coding RNA molecule may target aGA3 oxidase_2 gene for suppression and comprise a sequence that is atleast 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, at least 99.5%, or 100%complementary to at least 15, at least 16, at least 17, at least 18, atleast 19, at least 20, at least 21, at least 22, at least 23, at least24, at least 25, at least 26, or at least 27 consecutive nucleotides ofSEQ ID NO: 31 or SEQ ID NO: 32. According to some embodiments, anon-coding RNA molecule targeting the GA3 oxidase gene for suppressionmay be complementary to at least 19 consecutive nucleotides, but no morethan 27 consecutive nucleotides, such as complementary to 19, 20, 21,22, 23, 24, 25, 26, or 27 consecutive nucleotides, of SEQ ID NO: 31 orSEQ ID NO: 32. According to some embodiments, a non-coding RNA moleculetargeting the GA3 oxidase gene for suppression comprises a sequence thatis at least 80%, at least 85%, at least 90%, at least 95%, at least 96%,at least 97%, at least 98%, at least 99%, at least 99.5%, or 100%complementary to at least 15, at least 16, at least 17, at least 18, atleast 19, at least 20, at least 21, at least 22, at least 23, at least24, at least 25, at least 26, or at least 27 consecutive nucleotides ofa mRNA molecule encoding an endogenous GA3 oxidase protein in the plantthat is at least 80%, at least 85%, at least 90%, at least 95%, at least96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100%identical to SEQ ID NO: 33. According to further embodiments, anon-coding RNA molecule may comprise a sequence that is at least 80%, atleast 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, at least 99.5%, or 100% complementary to atleast 15, at least 16, at least 17, at least 18, at least 19, at least20, at least 21, at least 22, at least 23, at least 24, at least 25, atleast 26, or at least 27 consecutive nucleotides of a mRNA moleculeencoding an endogenous GA3 oxidase protein that is at least 80%, atleast 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, at least 99.5%, or 100% similar to SEQ ID NO:33.

As mentioned above, a non-coding RNA molecule may target an intronsequence of a GA3 oxidase gene instead of, or in addition to, an exonic,5′ UTR or 3′ UTR of the GA3 oxidase gene. Thus, a non-coding RNAmolecule targeting the GA3 oxidase_2 gene for suppression may comprise asequence that is at least 80%, at least 85%, at least 90%, at least 95%,at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%,or 100% complementary to at least 15, at least 16, at least 17, at least18, at least 19, at least 20, at least 21, at least 22, at least 23, atleast 24, at least 25, at least 26, or at least 27 consecutivenucleotides of SEQ ID NO: 37, and/or of nucleotides 533-692 or 852-982of SEQ ID NO: 37. The sequences provided herein for GA3 oxidase_2 mayvary across the diversity of corn plants, lines and germplasms due topolymorphisms and/or the presence of different alleles of the gene.Furthermore, a GA3 oxidase_2 gene may be expressed as alternativelyspliced isoforms that may give rise to different mRNA, cDNA and codingsequences that can affect the design of a suppression construct andnon-coding RNA molecule. Thus, a non-coding RNA molecule targeting a GA3oxidase_2 gene for suppression may be defined more broadly as comprisinga sequence that is at least 80%, at least 85%, at least 90%, at least95%, at least 96%, at least 97%, at least 98%, at least 99%, at least99.5%, or 100% complementary to at least 15, at least 16, at least 17,at least 18, at least 19, at least 20, at least 21, at least 22, atleast 23, at least 24, at least 25, at least 26, or at least 27consecutive nucleotides of SEQ ID NO: 37.

According to particular embodiments, a non-coding RNA molecule encodedby a transcribable DNA sequence for targeting a GA3 oxidase genecomprises (i) a sequence that is at least 95%, at least 96%, at least97%, at least 98%, at least 99%, at least 99.5%, or 100% complementaryto SEQ ID NO: 57 or 59, and/or (ii) a sequence or suppression elementencoding a non-coding RNA molecule comprising a sequence that is atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, atleast 99.5%, or 100% identical to SEQ ID NO: 58 or 60. According to someembodiments, the non-coding RNA molecule encoded by a transcribable DNAsequence may comprise a sequence with one or more mismatches, such as 1,2, 3, 4, 5 or more complementary mismatches, relative to the sequence ofa target or recognition site of a targeted GA3 oxidase gene mRNA, suchas a sequence that is nearly complementary to SEQ ID NO: 57 or 59 butwith one or more complementary mismatches relative to SEQ ID NO: 57 or59. According to a particular embodiment, the non-coding RNA moleculeencoded by the transcribable DNA sequence comprises a sequence that is100% identical to SEQ ID NO: 58 or 60, which is 100% complementary to atarget sequence within the cDNA and coding sequences of a GA3 oxidase_1or GA3 oxidase_2 gene in corn (i.e., SEQ ID NOs: 28, 29, 31 and/or 32),and/or to a corresponding sequence of a mRNA encoded by an endogenousGA3 oxidase_1 or GA3 oxidase_2 gene.

According to some embodiments, a non-coding RNA molecule may target aGA20 oxidase_4 gene for suppression and comprise a sequence that is atleast 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, at least 99.5%, or 100%complementary to at least 15, at least 16, at least 17, at least 18, atleast 19, at least 20, at least 21, at least 22, at least 23, at least24, at least 25, at least 26, or at least 27 consecutive nucleotides ofSEQ ID NO: 10 or SEQ ID NO: 11. According to some embodiments, anon-coding RNA molecule targeting a GA20 oxidase_4 gene for suppressionmay be complementary to at least 19 consecutive nucleotides, but no morethan 27 consecutive nucleotides, such as complementary to 19, 20, 21,22, 23, 24, 25, 26, or 27 consecutive nucleotides, of SEQ ID NO: 10 orSEQ ID NO: 11. According to some embodiments, a non-coding RNA moleculetargeting the GA20 oxidase gene for suppression comprises a sequencethat is at least 80%, at least 85%, at least 90%, at least 95%, at least96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100%complementary to at least 15, at least 16, at least 17, at least 18, atleast 19, at least 20, at least 21, at least 22, at least 23, at least24, at least 25, at least 26, or at least 27 consecutive nucleotides ofa mRNA molecule encoding an endogenous GA20 oxidase protein in the plantthat is at least 80%, at least 85%, at least 90%, at least 95%, at least96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100%identical to SEQ ID NO: 12. According to further embodiments, anon-coding RNA molecule may comprise a sequence that is at least 80%, atleast 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, at least 99.5%, or 100% complementary to atleast 15, at least 16, at least 17, at least 18, at least 19, at least20, at least 21, at least 22, at least 23, at least 24, at least 25, atleast 26, or at least 27 consecutive nucleotides of a mRNA moleculeencoding an endogenous GA20 oxidase protein that is at least 80%, atleast 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, at least 99.5%, or 100% similar to SEQ ID NO:12.

As mentioned above, a non-coding RNA molecule may target an intronsequence of a GA20 oxidase gene instead of, or in addition to, anexonic, 5′ UTR or 3′ UTR of the GA20 oxidase gene. Thus, a non-codingRNA molecule targeting a GA20 oxidase_4 gene for suppression maycomprise a sequence that is at least 80%, at least 85%, at least 90%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, atleast 99.5%, or 100% complementary to at least 15, at least 16, at least17, at least 18, at least 19, at least 20, at least 21, at least 22, atleast 23, at least 24, at least 25, at least 26, or at least 27consecutive nucleotides of SEQ ID NO: 38, and/or of nucleotides1996-2083 or 2412-2516 of SEQ ID NO: 38. The sequences provided hereinfor GA20 oxidase_4 may vary across the diversity of corn plants, linesand germplasms due to polymorphisms and/or the presence of differentalleles of the gene. Furthermore, a GA20 oxidase_4 gene may be expressedas alternatively spliced isoforms that may give rise to different mRNA,cDNA and coding sequences that can affect the design of a suppressionconstruct and non-coding RNA molecule. Thus, a non-coding RNA moleculetargeting a GA20 oxidase_4 gene for suppression may be defined morebroadly as comprising a sequence that is at least 80%, at least 85%, atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, at least 99.5%, or 100% complementary to at least 15, atleast 16, at least 17, at least 18, at least 19, at least 20, at least21, at least 22, at least 23, at least 24, at least 25, at least 26, orat least 27 consecutive nucleotides of SEQ ID NO: 38.

According to particular embodiments, a non-coding RNA molecule encodedby a transcribable DNA sequence for targeting a GA20 oxidase_4 genecomprises (i) a sequence that is at least 95%, at least 96%, at least97%, at least 98%, at least 99%, at least 99.5%, or 100% complementaryto SEQ ID NO: 61, and/or (ii) a sequence or suppression element encodinga non-coding RNA molecule comprising a sequence that is at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or100% identical to SEQ ID NO: 62. According to some embodiments, thenon-coding RNA molecule encoded by a transcribable DNA sequence maycomprise a sequence with one or more mismatches, such as 1, 2, 3, 4, 5or more complementary mismatches, relative to the sequence of a targetor recognition site of a targeted GA20 oxidase gene mRNA, such as asequence that is nearly complementary to SEQ ID NO: 61 but with one ormore complementary mismatches relative to SEQ ID NO: 61. According to aparticular embodiment, the non-coding RNA molecule encoded by thetranscribable DNA sequence comprises a sequence that is 100% identicalto SEQ ID NO: 62, which is 100% complementary to a target sequencewithin the cDNA and coding sequences of a GA20 oxidase_4 gene in corn(i.e., SEQ ID NO: 10 or 11), and/or to a corresponding sequence of amRNA encoded by an endogenous GA20 oxidase_4 gene.

According to embodiments of the present disclosure, a recombinant DNAconstruct is provided comprising a transcribable DNA sequence encoding anon-coding RNA molecule targeting an endogenous GA20 oxidase_3 and/orthe GA20 oxidase_5 gene(s) for suppression, wherein the transcribableDNA sequence is operably linked to a constitutive, tissue-specific ortissue-preferred promoter, and wherein the transcribable DNA sequencecauses the expression level of an endogenous GA20 oxidase_3 and/or theGA20 oxidase_5 gene(s) to become reduced or lowered in one or moretissue(s) of a plant transformed with the transcribable DNA sequence.Such a non-coding RNA molecule encoded by the transcribable DNA sequencemay comprise a sequence that is (i) at least 80%, at least 85%, at least90%, at least 95%, at least 96%, at least 97%, at least 98%, at least99%, at least 99.5%, or 100% complementary to at least 15, at least 16,at least 17, at least 18, at least 19, at least 20, at least 21, atleast 22, at least 23, at least 24, at least 25, at least 26, or atleast 27 consecutive nucleotides of a mRNA molecule encoding anendogenous GA20 oxidase protein in the plant that is at least 80%, atleast 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO:9, and/or (ii) at least 80%, at least 85%, at least 90%, at least 95%,at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%,or 100% complementary to at least 15, at least 16, at least 17, at least18, at least 19, at least 20, at least 21, at least 22, at least 23, atleast 24, at least 25, at least 26, or at least 27 consecutivenucleotides of a mRNA molecule encoding an endogenous GA20 oxidaseprotein in the plant that is at least 80%, at least 85%, at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, atleast 99.5%, or 100% identical to SEQ ID NO: 15.

According to embodiments of the present disclosure, a recombinant DNAconstruct is provided comprising a transcribable DNA sequence encoding anon-coding RNA molecule targeting an endogenous GA3 oxidase_1 and/or theGA3 oxidase_2 gene(s) for suppression, wherein the transcribable DNAsequence is operably linked to a constitutive, tissue-specific ortissue-preferred promoter, and wherein the transcribable DNA sequencecauses the expression level of an endogenous GA3 oxidase_1 and/or theGA3 oxidase_2 gene(s) to become reduced or lowered in one or moretissue(s) of a plant transformed with the transcribable DNA sequence.

Such a non-coding RNA molecule encoded by the transcribable DNA sequencemay comprise a sequence that is (i) at least 80%, at least 85%, at least90%, at least 95%, at least 96%, at least 97%, at least 98%, at least99%, at least 99.5%, or 100% complementary to at least 15, at least 16,at least 17, at least 18, at least 19, at least 20, at least 21, atleast 22, at least 23, at least 24, at least 25, at least 26, or atleast 27 consecutive nucleotides of a mRNA molecule encoding anendogenous GA3 oxidase protein in the plant that is at least 80%, atleast 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO:30, and/or (ii) at least 80%, at least 85%, at least 90%, at least 95%,at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%,or 100% complementary to at least 15, at least 16, at least 17, at least18, at least 19, at least 20, at least 21, at least 22, at least 23, atleast 24, at least 25, at least 26, or at least 27 consecutivenucleotides of a mRNA molecule encoding an endogenous GA3 oxidaseprotein in the plant that is at least 80%, at least 85%, at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, atleast 99.5%, or 100% identical to SEQ ID NO: 33.

According to embodiments of the present disclosure, a recombinant DNAconstruct is provided comprising a transcribable DNA sequence encoding anon-coding RNA molecule targeting an endogenous GA20 oxidase_4 gene forsuppression, wherein the transcribable DNA sequence is operably linkedto a constitutive, tissue-specific or tissue-preferred promoter, andwherein the transcribable DNA sequence causes the expression level of anendogenous GA20 oxidase_4 gene to become reduced or lowered in one ormore tissue(s) of a plant transformed with the transcribable DNAsequence. Such a non-coding RNA molecule encoded by the transcribableDNA sequence may comprise a sequence that is (i) at least 80%, at least85%, at least 90%, at least 95%, at least 96%, at least 97%, at least98%, at least 99%, at least 99.5%, or 100% complementary to at least 15,at least 16, at least 17, at least 18, at least 19, at least 20, atleast 21, at least 22, at least 23, at least 24, at least 25, at least26, or at least 27 consecutive nucleotides of a mRNA molecule encodingan endogenous GA20 oxidase protein in the plant that is at least 80%, atleast 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO:12.

According to many embodiments, a modified or transgenic plant isprovided that is transformed with a recombinant DNA construct comprisinga transcribable DNA sequence encoding a non-coding RNA moleculetargeting an endogenous GA20 oxidase_3 and/or GA20 oxidase_5 gene(s) forsuppression, and/or has an endogenous GA20 oxidase_3 and/or GA20oxidase_5 gene edited through targeted genome editing techniques, asprovided herein, wherein the transcribable DNA sequence is operablylinked to a constitutive promoter or a tissue-specific ortissue-preferred promoter, such as a vascular promoter or a leafpromoter, and wherein the expression level of the endogenous GA20oxidase_3 and/or GA20 oxidase_5 gene(s) is eliminated, reduced orlowered in one or more plant tissue(s), such as one or more vascularand/or leaf tissue(s), of the modified or transgenic plant by at least5%, at least 10%, at least 20%, at least 25%, at least 30%, at least40%, at least 50%, at least 60%, at least 70%, at least 75%, at least80%, at least 90%, or 100% as compared to a wild type or control plant.According to many embodiments, a modified or transgenic plant isprovided that is transformed with a recombinant DNA construct comprisinga transcribable DNA sequence encoding a non-coding RNA moleculetargeting an endogenous GA20 oxidase_3 and/or GA20 oxidase_5 gene(s) forsuppression, and/or has an endogenous GA20 oxidase_3 and/or GA20oxidase_5 gene(s) edited through targeted genome editing techniques toreduce or eliminate its level of expression and/or activity, wherein thetranscribable DNA sequence is operably linked to a constitutive promoteror a tissue-specific or tissue-preferred promoter, such as a vascularpromoter or a leaf promoter, and wherein the level of one or more activeGAs, such as GA1, GA3, GA4, and/or GA7, is reduced or lowered in one ormore plant tissue(s), such as one or more stem, internode, vascularand/or leaf tissue(s) or one or more stem and/or internode tissue(s), ofthe modified or transgenic plant by at least 5%, at least 10%, at least20%, at least 25%, at least 30%, at least 40%, at least 50%, at least60%, at least 70%, at least 75%, at least 80%, at least 90%, or 100% ascompared to a wild type or control plant.

According to many embodiments, a modified or transgenic plant isprovided that is transformed with a recombinant DNA construct comprisinga transcribable DNA sequence encoding a non-coding RNA moleculetargeting an endogenous GA3 oxidase_1 and/or GA3 oxidase_2 gene(s) forsuppression, and/or has an endogenous GA3 oxidase_1 or GA3 oxidase_2gene edited through targeted genome editing techniques, as providedherein, wherein the transcribable DNA sequence is operably linked to aconstitutive promoter or a tissue-specific or tissue-preferred promoter,such as a vascular promoter or a leaf promoter, and wherein theexpression level of the endogenous GA3 oxidase_1 and/or GA3 oxidase_2gene(s) is eliminated, reduced or lowered in one or more planttissue(s), such as one or more vascular and/or leaf tissue(s), of themodified or transgenic plant by at least 5%, at least 10%, at least 20%,at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, atleast 70%, at least 75%, at least 80%, at least 90%, or 100% as comparedto a wild type or control plant. According to many embodiments, amodified or transgenic plant is provided that is transformed with arecombinant DNA construct comprising a transcribable DNA sequenceencoding a non-coding RNA molecule targeting an endogenous GA3 oxidase_1and/or GA3 oxidase_2 gene(s) for suppression, and/or has an endogenousGA3 oxidase_1 and/or GA3 oxidase_2 gene edited through targeted genomeediting techniques to reduce or eliminate its level of expression and/oractivity, wherein the transcribable DNA sequence is operably linked to aconstitutive promoter or a tissue-specific or tissue-preferred promoter,such as a vascular promoter or a leaf promoter, and wherein the level ofone or more active GAs, such as GA1, GA3, GA4, and/or GA7, is reduced orlowered in one or more plant tissue(s), such as one or more stem,internode, vascular and/or leaf tissue(s) or one or more stem and/orinternode tissue(s), of the modified or transgenic plant by at least 5%,at least 10%, at least 20%, at least 25%, at least 30%, at least 40%, atleast 50%, at least 60%, at least 70%, at least 75%, at least 80%, atleast 90%, or 100% as compared to a wild type or control plant.

According to many embodiments, a modified or transgenic plant isprovided that is transformed with a recombinant DNA construct comprisinga transcribable DNA sequence encoding a non-coding RNA moleculetargeting an endogenous GA20 oxidase_4 gene for suppression, and/or hasan endogenous GA20 oxidase_4 gene edited through targeted genome editingtechniques, as provided herein, wherein the transcribable DNA sequenceis operably linked to a constitutive promoter or a tissue-specific ortissue-preferred promoter, such as a vascular promoter or a leafpromoter, and wherein the expression level of the endogenous GA20oxidase_4 gene(s) is eliminated, reduced or lowered in one or more planttissue(s), such as one or more vascular and/or leaf tissue(s), of themodified or transgenic plant by at least 5%, at least 10%, at least 20%,at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, atleast 70%, at least 75%, at least 80%, at least 90%, or 100% as comparedto a wild type or control plant. According to many embodiments, amodified or transgenic plant is provided that is transformed with arecombinant DNA construct comprising a transcribable DNA sequenceencoding a non-coding RNA molecule targeting an endogenous GA20oxidase_4 gene(s) for suppression, and/or has an endogenous GA20oxidase_4 gene edited through targeted genome editing techniques toreduce or eliminate its level of expression and/or activity, wherein thetranscribable DNA sequence is operably linked to a constitutive promoteror a tissue-specific or tissue-preferred promoter, such as a vascularpromoter or a leaf promoter, and wherein the level of one or more activeGAs, such as GA1, GA3, GA4, and/or GA7, is reduced or lowered in one ormore plant tissue(s), such as one or more stem, internode, vascularand/or leaf tissue(s) or one or more stem and/or internode tissue(s), ofthe modified or transgenic plant by at least 5%, at least 10%, at least20%, at least 25%, at least 30%, at least 40%, at least 50%, at least60%, at least 70%, at least 75%, at least 80%, at least 90%, or 100% ascompared to a wild type or control plant.

According to many embodiments, a modified or transgenic plant isprovided that is transformed with a recombinant DNA construct comprisinga transcribable DNA sequence encoding a non-coding RNA moleculetargeting an endogenous GA20 oxidase_3, GA20 oxidase_4, and/or GA20oxidase_5 gene(s) for suppression, is transformed with a recombinant DNAconstruct comprising a transcribable DNA sequence encoding a non-codingRNA molecule targeting an endogenous GA3 oxidase_1 and/or the GA3oxidase_2 gene(s) for suppression, and/or has an endogenous GA20oxidase_3, GA20 oxidase_4, or the GA20 oxidase_5 gene edited throughtargeted genome editing techniques, to reduce or eliminate its level ofexpression and/or activity, as provided herein, wherein thetranscribable DNA sequence is operably linked to a constitutive promoteror a tissue-specific or tissue-preferred promoter, such as a vascularpromoter or a leaf promoter, and wherein the modified or transgenicplant has one or more of the following traits: a semi-dwarf or reducedplant height or stature, decreased stem internode length, increasedlodging resistance, and/or increased stem or stalk diameter. Such amodified or transgenic plant may not have any significant reproductiveoff-types. A modified or transgenic plant may have one or more of thefollowing additional traits: reduced green snap, deeper roots, increasedleaf area, earlier canopy closure, higher stomatal conductance, lowerear height, increased foliar water content, improved drought tolerance,increased nitrogen use efficiency, increased water use efficiency,reduced anthocyanin content and anthocyanin area in leaves under normaland/or nitrogen or water limiting stress conditions, increased earweight, increased kernel number, increased kernel weight, increasedyield, and/or increased harvest index. According to many of theseembodiments, the level of expression and/or activity of an endogenousGA20 oxidase_3, GA20 oxidase_4, and/or GA20 oxidase_5 gene(s), or anendogenous GA3 oxidase_1 and/or GA3 oxidase_2 gene(s), may beeliminated, reduced or lowered in one or more plant tissue(s), such asone or more vascular and/or leaf tissue(s), of the modified ortransgenic plant by at least 5%, at least 10%, at least 20%, at least25%, at least 30%, at least 40%, at least 50%, at least 60%, at least70%, at least 75%, at least 80%, at least 90%, or 100% as compared to awild type or control plant, and/or the level of one or more active GAs,such as GA1, GA3, GA4, and/or GA7, is reduced or lowered in one or moreplant tissue(s), such as one or more stem, internode, vascular and/orleaf tissue(s), or one or more stem and/or internode tissue(s), of themodified or transgenic plant by at least 5%, at least 10%, at least 20%,at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, atleast 70%, at least 75%, at least 80%, at least 90%, or 100% as comparedto a wild type or control plant.

According to many of the embodiments described in the above paragraphs,the non-coding RNA molecule encoded by the transcribable DNA sequence ofthe recombinant DNA molecule, vector or construct may be a precursormiRNA or siRNA that may be subsequently processed or cleaved in a plantcell to form a mature miRNA or siRNA.

A recombinant DNA molecule, construct or vector of the presentdisclosure may comprise a transcribable DNA sequence encoding anon-coding RNA molecule that targets an endogenous GA oxidase gene forsuppression, wherein the transcribable DNA sequence is operativelylinked to a plant-expressible promoter, such as a constitutive orvascular and/or leaf promoter. For purposes of the present disclosure, anon-coding RNA molecule encoded by a transcribable DNA sequence thattargets an endogenous GA oxidase gene for suppression may include amature non-coding RNA molecule that targets an endogenous GA oxidasegene for suppression, and/or a precursor RNA molecule that may becomeprocessed in a plant cell into a mature non-coding RNA molecule, such asa miRNA or siRNA, that targets an endogenous GA oxidase gene forsuppression. In addition to its associated promoter, a transcribable DNAsequence encoding a non-coding RNA molecule for suppression of anendogenous GA oxidase gene may also be operatively linked to one or moreadditional regulatory element(s), such as an enhancer(s), leader,transcription start site (TSS), linker, 5′ and 3′ untranslated region(s)(UTRs), intron(s), polyadenylation signal, termination region orsequence, etc., that are suitable, necessary or preferred forstrengthening, regulating or allowing expression of the transcribableDNA sequence in a plant cell. Such additional regulatory element(s) maybe optional and/or used to enhance or optimize expression of thetransgene or transcribable DNA sequence. As provided herein, an“enhancer” may be distinguished from a “promoter” in that an enhancertypically lacks a transcription start site, TATA box, or equivalentsequence and is thus insufficient alone to drive transcription. As usedherein, a “leader” may be defined generally as the DNA sequence of the5′-UTR of a gene (or transgene) between the transcription start site(TSS) and 5′ end of the transcribable DNA sequence or protein codingsequence start site of the transgene.

According to further embodiments, methods are provided for transforminga plant cell, tissue or explant with a recombinant DNA molecule orconstruct comprising a transcribable DNA sequence or transgene operablylinked to a plant-expressible promoter to produce a transgenic plant.The transcribable DNA sequence may encode a non-coding RNA molecule thattargets a GA oxidase gene(s) for suppression, or a RNA precursor that isprocessed into a mature RNA molecule, such as a miRNA or siRNA, thattargets one or more GA oxidase gene(s) for suppression. Numerous methodsfor transforming chromosomes or plastids in a plant cell with arecombinant DNA molecule or construct are known in the art, which may beused according to method embodiments of the present invention to producea transgenic plant cell and plant. Any suitable method or technique fortransformation of a plant cell known in the art may be used according topresent methods. Effective methods for transformation of plants includebacterially mediated transformation, such as Agrobacterium-mediated orRhizobium-mediated transformation, and microprojectile or particlebombardment-mediated transformation. A variety of methods are known inthe art for transforming explants with a transformation vector viabacterially mediated transformation or microprojectile or particlebombardment and then subsequently culturing, etc., those explants toregenerate or develop transgenic plants. Other methods for planttransformation, such as microinjection, electroporation, vacuuminfiltration, pressure, sonication, silicon carbide fiber agitation,PEG-mediated transformation, etc., are also known in the art.

Methods of transforming plant cells and explants are well known bypersons of ordinary skill in the art. Methods for transforming plantcells by microprojectile bombardment with particles coated withrecombinant DNA are provided, for example, in U.S. Pat. Nos. 5,550,318;5,538,880 6,160,208; 6,399,861; and 6,153,812, andAgrobacterium-mediated transformation is described, for example, in U.S.Pat. Nos. 5,159,135; 5,824,877; 5,591,616; 6,384,301; 5,750,871;5,463,174; and 5,188,958, all of which are incorporated herein byreference. Additional methods for transforming plants can be found in,for example, Compendium of Transgenic Crop Plants (2009) BlackwellPublishing. Any suitable method of plant transformation known or laterdeveloped in the art can be used to transform a plant cell or explantwith any of the nucleic acid molecules, constructs or vectors providedherein.

Transgenic plants produced by transformation methods may be chimeric ornon-chimeric for the transformation event depending on the methods andexplants used. Methods are further provided for expressing a non-codingRNA molecule that targets an endogenous GA oxidase gene for suppressionin one or more plant cells or tissues under the control of aplant-expressible promoter, such as a constitutive, tissue-specific,tissue-preferred, vascular and/or leaf promoter as provided herein. Suchmethods may be used to create transgenic cereal or corn plants having ashorter, semi-dwarf stature, reduced internode length, increasedstalk/stem diameter, and/or improved lodging resistance. Such transgeniccereal or corn plants may further have other traits that may bebeneficial for yield, such as reduced green snap, deeper roots,increased leaf area, earlier canopy closure, improved drought tolerance,increased nitrogen use efficiency, increased water use efficiency,higher stomatal conductance, lower ear height, increased foliar watercontent, reduced anthocyanin content and/or area in leaves under normalor nitrogen or water limiting stress conditions, increased ear weight,increased seed or kernel number, increased seed or kernel weight,increased yield, and/or increased harvest index, relative to a wild typeor control plant. As used herein, “harvest index” refers to the mass ofthe harvested grain divided by the total mass of the above-groundbiomass of the plant over a harvested area.

Transgenic plants expressing a GA oxidase transgene or non-coding RNAmolecule that targets an endogenous GA oxidase gene for suppression mayhave an earlier canopy closure (e.g., approximately one day earlier, or12-48 hours, 12-36 hours, 18-36 hours, or about 24 hours earlier canopyclosure) than a wild type or control plant. Although transgenic plantsexpressing a GA oxidase transgene or non-coding RNA molecule thattargets an endogenous GA oxidase gene for suppression may have a lowerear height than a wild type or control plant, the height of the ear maygenerally be at least 18 inches above the ground. Transgenic plantsexpressing a non-coding RNA molecule that targets an endogenous GAoxidase gene for suppression may have greater biomass and/or leaf areaduring one or more late vegetative stages (e.g., V8-V12) than a wildtype or control plant. Transgenic plants expressing a GA oxidasetransgene or non-coding RNA molecule that targets an endogenous GAoxidase gene for suppression may have deeper roots during latervegetative stages when grown in the field, than a wild type or controlplant, which may be due to an increased root front velocity. Thesetransgenic plants may reach a depth 90 cm below ground sooner (e.g.,10-25 days sooner, 15-25 days sooner, or about 20 days sooner) than awild type or control plant, which may occur by the vegetative toreproductive transition of the plant (e.g., by V16/R1 at about 50 daysafter planting as opposed at about 70 days after planting for controlplants).

Recipient cell(s) or explant or cellular targets for transformationinclude, but are not limited to, a seed cell, a fruit cell, a leaf cell,a cotyledon cell, a hypocotyl cell, a meristem cell, an embryo cell, anendosperm cell, a root cell, a shoot cell, a stem cell, a pod cell, aflower cell, an inflorescence cell, a stalk cell, a pedicel cell, astyle cell, a stigma cell, a receptacle cell, a petal cell, a sepalcell, a pollen cell, an anther cell, a filament cell, an ovary cell, anovule cell, a pericarp cell, a phloem cell, a bud cell, a callus cell, achloroplast, a stomatal cell, a trichome cell, a root hair cell, astorage root cell, or a vascular tissue cell, a seed, embryo, meristem,cotyledon, hypocotyl, endosperm, root, shoot, stem, node, callus, cellsuspension, protoplast, flower, leaf, pollen, anther, ovary, ovule,pericarp, bud, and/or vascular tissue, or any transformable portion ofany of the foregoing. For plant transformation, any target cell(s),tissue(s), explant(s), etc., that may be used to receive a recombinantDNA transformation vector or molecule of the present disclosure may becollectively be referred to as an “explant” for transformation.Preferably, a transformable or transformed explant cell or tissue may befurther developed or regenerated into a plant. Any cell or explant fromwhich a fertile plant can be grown or regenerated is contemplated as auseful recipient cell or explant for practice of this disclosure (i.e.,as a target explant for transformation). Callus can be initiated orcreated from various tissue sources, including, but not limited to,embryos or parts of embryos, non-embryonic seed tissues, seedling apicalmeristems, microspores, and the like. Any cells that are capable ofproliferating as callus may serve as recipient cells for transformation.Transformation methods and materials for making transgenic plants (e.g.,various media and recipient target cells or explants and methods oftransformation and subsequent regeneration of into transgenic plants)are known in the art.

Transformation of a target plant material or explant may be practiced intissue culture on nutrient media, for example a mixture of nutrientsthat allow cells to grow in vitro or cell culture. Transformed explants,cells or tissues may be subjected to additional culturing steps, such ascallus induction, selection, regeneration, etc., as known in the art.Transformation may also be carried out without creation or use of acallus tissue. Transformed cells, tissues or explants containing arecombinant DNA sequence insertion or event may be grown, developed orregenerated into transgenic plants in culture, plugs, or soil accordingto methods known in the art. Transgenic plants may be further crossed tothemselves or other plants to produce transgenic seeds and progeny. Atransgenic plant may also be prepared by crossing a first plantcomprising the recombinant DNA sequence or transformation event with asecond plant lacking the insertion. For example, a recombinant DNAconstruct or sequence may be introduced into a first plant line that isamenable to transformation, which may then be crossed with a secondplant line to introgress the recombinant DNA construct or sequence intothe second plant line. Progeny of these crosses can be further backcrossed into the more desirable line multiple times, such as through 6to 8 generations or back crosses, to produce a progeny plant withsubstantially the same genotype as the original parental line, but forthe introduction of the recombinant DNA construct or sequence.

A transgenic or edited plant, plant part, cell, or explant providedherein may be of an elite variety or an elite line. An elite variety oran elite line refers to a variety that has resulted from breeding andselection for superior agronomic performance. A transgenic or editedplant, cell, or explant provided herein may be a hybrid plant, cell, orexplant. As used herein, a “hybrid” is created by crossing two plantsfrom different varieties, lines, inbreds, or species, such that theprogeny comprises genetic material from each parent. Skilled artisansrecognize that higher order hybrids can be generated as well. Forexample, a first hybrid can be made by crossing Variety A with Variety Bto create a A×B hybrid, and a second hybrid can be made by crossingVariety C with Variety D to create an C×D hybrid. The first and secondhybrids can be further crossed to create the higher order hybrid(A×B)×(C×D) comprising genetic information from all four parentvarieties.

According to embodiments of the present disclosure, a modified plant isprovided comprising a GA oxidase suppression element that targets two ormore GA oxidase genes for suppression, or a combination of two or moreGA oxidase suppression element(s) and/or gene edit(s). A recombinant DNAconstruct or vector may comprise a single cassette or suppressionelement comprising a transcribable DNA sequence designed or chosen toencode a non-coding RNA molecule that is complementary to mRNArecognition or target sequences of two or more GA oxidase genesincluding at least a first GA oxidase gene and a second GA oxidasegene—i.e., the mRNAs of the targeted GA oxidase genes share an identicalor nearly identical (or similar) sequence such that a single suppressionelement and encoded non-coding RNA molecule can target each of thetargeted GA oxidase genes for suppression. For example, an expressioncassette and suppression construct is provided herein comprising atranscribable DNA sequence that encodes a single non-coding RNA moleculethat targets both the GA20 oxidase_3 and GA20 oxidase_5 genes forsuppression.

According to other embodiments, a recombinant DNA construct or vectormay comprise two or more suppression elements or sequences that may bestacked together in a construct or vector either in tandem in a singleexpression cassette or separately in two or more expression cassettes. Arecombinant DNA construct or vector may comprise a single expressioncassette or suppression element comprising a transcribable DNA sequencethat encodes a non-coding RNA molecule comprising two or more targetingsequences arranged in tandem, including at least a first targetingsequence and a second targeting sequence, wherein the first targetingsequence is complementary to a mRNA recognition or target site of afirst GA oxidase gene, and the second targeting sequence iscomplementary to a mRNA recognition or target site of a second GAoxidase gene, and wherein the transcribable DNA sequence is operablylinked to a plant-expressible promoter. The plant-expressible promotermay be a constitutive promoter, or a tissue-specific or tissue-preferredpromoter, as provided herein. The non-coding RNA molecule may beexpressed as a pre-miRNA that becomes processed into two or more maturemiRNAs including at least a first mature miRNA and a second miRNA,wherein the first miRNA comprises a targeting sequence that iscomplementary to the mRNA recognition or target site of the first GAoxidase gene, and the second miRNA comprises a targeting sequence thatis complementary to the mRNA recognition or target site of the second GAoxidase gene.

According to other embodiments, a recombinant DNA construct or vectormay comprise two or more expression cassettes including a firstexpression cassette and a second expression cassette, wherein the firstexpression cassette comprises a first transcribable DNA sequenceoperably linked to a first plant-expressible promoter, and the secondexpression cassette comprises a second transcribable DNA sequenceoperably linked to a second plant-expressible promoter, wherein thefirst transcribable DNA sequence encodes a first non-coding RNA moleculecomprising a targeting sequence that is complementary to a mRNArecognition or target site of a first GA oxidase gene, and the secondtranscribable DNA sequence encodes a second non-coding RNA moleculecomprising a targeting sequence that is complementary to a mRNArecognition or target site of a second GA oxidase gene. The first andsecond plant-expressible promoters may each be a constitutive promoter,or a tissue-specific or tissue-preferred promoter, as provided herein,and the first and second plant-expressible promoters may be the same ordifferent promoters.

According to other embodiments, two or more suppression elements orconstructs targeting GA oxidase gene(s) and/or GA oxidase gene edit(s)may be combined in a modified plant by crossing two or more plantstogether in one or more generations to produce a modified plant having adesired combination of suppression element(s) and/or gene edit(s).According to these embodiments, a first modified plant comprising asuppression element or construct targeting a GA oxidase gene(s) (or a GAoxidase gene edit) may be crossed to a second modified plant comprisinga suppression element or construct targeting a GA oxidase gene(s) (or aGA oxidase gene edit), such that a modified progeny plant may be madecomprising a first suppression element or construct and a secondsuppression element or construct, a suppression element or construct anda GA oxidase gene edit, or a first GA oxidase gene edit and a second GAoxidase gene edit. Alternatively, a modified plant comprising two ormore suppression elements or constructs targeting GA oxidase gene(s)and/or GA oxidase gene edit(s) may be made by (i) co-transforming afirst suppression element or construct and a second suppression elementor construct (each targeting a GA oxidase gene for suppression), (ii)transforming a modified plant with a second suppression element orconstruct, wherein the modified plant already comprises a firstsuppression element or construct, (iii) transforming a modified plantwith a suppression element or construct, wherein the modified plantalready comprises an edited GA oxidase gene, (iv) transforming amodified plant with a construct(s) for making one or more edits in GAoxidase gene(s), wherein the modified plant already comprises asuppression element or construct, or (v) transforming with construct(s)for making two or more edits in GA oxidase gene(s).

According to embodiments of the present disclosure, modified plants areprovided comprising two or more constructs targeting GA oxidase gene(s)for suppression including a first recombinant DNA construct and a secondrecombinant DNA construct, wherein the first recombinant DNA constructcomprises a first transcribable DNA sequence encoding a first non-codingRNA molecule that is complementary to a mRNA recognition or targetsequence of a first GA oxidase gene, and the second recombinant DNAconstruct comprises a second transcribable DNA sequence encoding asecond non-coding RNA molecule that is complementary to a mRNArecognition or target sequence of a second GA oxidase gene. The firstand second recombinant DNA constructs may be stacked in a single vectorand transformed into a plant as a single event, or present in separatevectors or constructs that may be transformed as separate events.According to these embodiments, the first GA oxidase gene may be a GA20oxidase_3, GA20 oxidase_5, GA20 oxidase_4, GA3 oxidase_1, or GA3oxidase_2 gene, the first non-coding RNA molecule is complementary to arecognition or target sequence of an mRNA expressed from such GA oxidasegene, and the second GA oxidase gene may be a GA20 oxidase_3, GA20oxidase_5, GA20 oxidase_4, GA3 oxidase_1, or GA3 oxidase_2 gene.According to some embodiments, the first and second GA oxidase genes maybe the same or different GA oxidase gene(s). Alternatively, the secondGA oxidase gene may be another GA oxidase gene, such as a GA20oxidase_1, GA20 oxidase_2, GA20 oxidase_6, GA20 oxidase_7, GA20oxidase_8, or GA20 oxidase_9 gene, and the second non-coding RNAmolecule is complementary to a recognition or target sequence of an mRNAexpressed from such GA oxidase gene.

According to embodiments of the present disclosure, modified plants areprovided comprising a recombinant DNA construct targeting GA oxidasegenes for suppression comprising a transcribable DNA sequence encoding anon-coding RNA molecule that comprises two or more targeting sequencesarranged in tandem including at least a first targeting sequence that iscomplementary to a mRNA recognition or target sequence of a first GAoxidase gene and a second targeting sequence that is complementary to amRNA recognition or target sequence of a second GA oxidase gene. Thenon-coding RNA molecule may be expressed as a pre-miRNA that becomesprocessed into two or more mature miRNAs including at least a firstmature miRNA and a second miRNA, wherein the first miRNA comprises thefirst targeting sequence that is complementary to the mRNA recognitionor target site of the first GA oxidase gene, and the second miRNAcomprises the second targeting sequence that is complementary to themRNA recognition or target site of the second GA oxidase gene. Accordingto these embodiments, the first GA oxidase gene may be a GA20 oxidase_3,GA20 oxidase_5, GA20 oxidase_4, GA3 oxidase_1, or GA3 oxidase_2 gene,the first non-coding RNA molecule is complementary to a recognition ortarget sequence of an mRNA expressed from such GA oxidase gene, and thesecond GA oxidase gene may be a GA20 oxidase_3, GA20 oxidase_5, GA20oxidase_4, GA3 oxidase_1, or GA3 oxidase_2 gene. According to someembodiments, the first and second GA oxidase genes may be the same ordifferent GA oxidase gene(s). Alternatively, the second GA oxidase genemay be another GA oxidase gene, such as a GA20 oxidase_1, GA20oxidase_2, GA20 oxidase_6, GA20 oxidase_7, GA20 oxidase_8, or GA20oxidase_9 gene, and the second non-coding RNA molecule is complementaryto a recognition or target sequence of an mRNA expressed from such GAoxidase gene.

In the above stacking scenarios, and regardless of whether the targetingsequences are stacked in tandem in a single transcribable DNA sequence(or expression cassette) or in separate transcribable DNA sequences (orexpression cassettes), the second GA oxidase gene may be a GA oxidasegene other than a GA20 oxidase_3, GA20 oxidase_5, GA20 oxidase_4, GA3oxidase_1, or GA3 oxidase_2 gene, such as a GA20 oxidase_1, GA20oxidase_2, GA20 oxidase_6, GA20 oxidase_7, GA20 oxidase_8, or GA20oxidase_9 gene. According to these embodiments, the second targetingsequence of a non-coding RNA molecule may be at least 80%, at least 85%,at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, at least 99.5%, or 100% complementary to at least 15, atleast 16, at least 17, at least 18, at least 19, at least 20, at least21, at least 22, at least 23, at least 24, at least 25, at least 26, orat least 27 consecutive nucleotides of any one or more of SEQ ID NOs: 1,2, 4, 5, 16, 17, 19, 20, 22, 23, 25, and/or 26. According to someembodiments, the second targeting sequence of a non-coding RNA moleculemay be complementary to at least 19 consecutive nucleotides, but no morethan 27 consecutive nucleotides, such as complementary to 19, 20, 21,22, 23, 24, 25, 26, or 27 consecutive nucleotides, of any one or more ofSEQ ID NOs: 1, 2, 4, 5, 16, 17, 19, 20, 22, 23, 25, and/or 26. Accordingto some embodiments, the second targeting sequence of a non-coding RNAmolecule may be at least 80%, at least 85%, at least 90%, at least 95%,at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%,or 100% complementary to at least 15, at least 16, at least 17, at least18, at least 19, at least 20, at least 21, at least 22, at least 23, atleast 24, at least 25, at least 26, or at least 27 consecutivenucleotides of a mRNA molecule encoding an endogenous GA oxidase proteinin the plant that is at least 80%, at least 85%, at least 90%, at least95%, at least 96%, at least 97%, at least 98%, at least 99%, at least99.5%, or 100% identical to any one or more of SEQ ID NOs: 3, 6, 18, 21,24, and/or 27. According to further embodiments, the second targetingsequence of a non-coding RNA molecule may comprise a sequence that is atleast 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, at least 99.5%, or 100%complementary to at least 15, at least 16, at least 17, at least 18, atleast 19, at least 20, at least 21, at least 22, at least 23, at least24, at least 25, at least 26, or at least 27 consecutive nucleotides ofa mRNA molecule encoding an endogenous GA oxidase protein that is atleast 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, at least 99.5%, or 100% similarto any one or more of SEQ ID NO: 3, 6, 18, 21, 24, and/or 27.

A recombinant DNA molecule or construct of the present disclosure maycomprise or be included within a DNA transformation vector for use intransformation of a target plant cell, tissue or explant. Such atransformation vector may generally comprise sequences or elementsnecessary or beneficial for effective transformation in addition to atleast one transgene, expression cassette and/or transcribable DNAsequence encoding a GA oxidase gene or a non-coding RNA moleculetargeting an endogenous GA oxidase gene for suppression. ForAgrobacterium-mediated, Rhizobia-mediated or other bacteria-mediatedtransformation, the transformation vector may comprise an engineeredtransfer DNA (or T-DNA) segment or region having two border sequences, aleft border (LB) and a right border (RB), flanking at least atranscribable DNA sequence or transgene, such that insertion of theT-DNA into the plant genome will create a transformation event for thetranscribable DNA sequence, transgene or expression cassette. Thus, atranscribable DNA sequence, transgene or expression cassette encoding anon-coding RNA molecule targeting an endogenous GA oxidase gene forsuppression may be located between the left and right borders of theT-DNA, perhaps along with an additional transgene(s) or expressioncassette(s), such as a plant selectable marker transgene and/or othergene(s) of agronomic interest that may confer a trait or phenotype ofagronomic interest to a plant. According to alternative embodiments, thetranscribable DNA sequence, transgene or expression cassette encoding anon-coding RNA molecule targeting an endogenous GA oxidase gene forsuppression and the plant selectable marker transgene (or other gene ofagronomic interest) may be present in separate T-DNA segments on thesame or different recombinant DNA molecule(s), such as forco-transformation. A transformation vector or construct may furthercomprise prokaryotic maintenance elements, which may be located in thevector outside of the T-DNA region(s).

A plant selectable marker transgene in a transformation vector orconstruct of the present disclosure may be used to assist in theselection of transformed cells or tissue due to the presence of aselection agent, such as an antibiotic or herbicide, wherein the plantselectable marker transgene provides tolerance or resistance to theselection agent. Thus, the selection agent may bias or favor thesurvival, development, growth, proliferation, etc., of transformed cellsexpressing the plant selectable marker gene, such as to increase theproportion of transformed cells or tissues in the R₀ plant. Commonlyused plant selectable marker genes include, for example, thoseconferring tolerance or resistance to antibiotics, such as kanamycin andparomomycin (nptII), hygromycin B (aph IV), streptomycin orspectinomycin (aadA) and gentamycin (aac3 and aacC4), or thoseconferring tolerance or resistance to herbicides such as glufosinate(bar or pat), dicamba (DMO) and glyphosate (aroA or EPSPS). Plantscreenable marker genes may also be used, which provide an ability tovisually screen for transformants, such as luciferase or greenfluorescent protein (GFP), or a gene expressing a beta glucuronidase oruidA gene (GUS) for which various chromogenic substrates are known. Insome embodiments, a vector or polynucleotide provided herein comprisesat least one selectable marker gene selected from the group consistingof nptll, aph IV, aadA, aac3, aacC4, bar, pat, DMO, EPSPS, aroA, GFP,and GUS. Plant transformation may also be carried out in the absence ofselection during one or more steps or stages of culturing, developing orregenerating transformed explants, tissues, plants and/or plant parts.

According to present embodiments, methods for transforming a plant cell,tissue or explant with a recombinant DNA molecule or construct mayfurther include site-directed or targeted integration. According tothese methods, a portion of a recombinant DNA donor template molecule(i.e., an insertion sequence) may be inserted or integrated at a desiredsite or locus within the plant genome. The insertion sequence of thedonor template may comprise a transgene or construct, such as atransgene or transcribable DNA sequence encoding a non-coding RNAmolecule that targets an endogenous GA oxidase gene for suppression. Thedonor template may also have one or two homology arms flanking theinsertion sequence to promote the targeted insertion event throughhomologous recombination and/or homology-directed repair. Each homologyarm may be at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 96%, at least 97%, at least 99% or100% identical or complementary to at least 20, at least 25, at least30, at least 35, at least 40, at least 45, at least 50, at least 60, atleast 70, at least 80, at least 90, at least 100, at least 150, at least200, at least 250, at least 500, at least 1000, at least 2500, or atleast 5000 consecutive nucleotides of a target DNA sequence within thegenome of a monocot or cereal plant. Thus, a recombinant DNA molecule ofthe present disclosure may comprise a donor template for site-directedor targeted integration of a transgene or construct, such as a transgeneor transcribable DNA sequence encoding a non-coding RNA molecule thattargets an endogenous GA oxidase gene for suppression, into the genomeof a plant.

Any site or locus within the genome of a plant may potentially be chosenfor site-directed integration of a transgene, construct or transcribableDNA sequence provided herein. For site-directed integration, adouble-strand break (DSB) or nick may first be made at a selectedgenomic locus with a site-specific nuclease, such as, for example, azinc-finger nuclease, an engineered or native meganuclease, aTALE-endonuclease, or an RNA-guided endonuclease (e.g., Cas9 or Cpf1).Any method known in the art for site-directed integration may be used.In the presence of a donor template molecule with an insertion sequence,the DSB or nick may then be repaired by homologous recombination betweenhomology arm(s) of the donor template and the plant genome, or bynon-homologous end joining (NHEJ), resulting in site-directedintegration of the insertion sequence into the plant genome to createthe targeted insertion event at the site of the DSB or nick. Thus,site-specific insertion or integration of a transgene, construct orsequence may be achieved.

The introduction of a DSB or nick may also be used to introduce targetedmutations in the genome of a plant. According to this approach,mutations, such as deletions, insertions, inversions and/orsubstitutions may be introduced at a target site via imperfect repair ofthe DSB or nick to produce a knock-out or knock-down of a GA oxidasegene. Such mutations may be generated by imperfect repair of thetargeted locus even without the use of a donor template molecule. A“knock-out” of a GA oxidase gene may be achieved by inducing a DSB ornick at or near the endogenous locus of the GA oxidase gene that resultsin non-expression of the GA oxidase protein or expression of anon-functional protein, whereas a “knock-down” of a GA oxidase gene maybe achieved in a similar manner by inducing a DSB or nick at or near theendogenous locus of the GA oxidase gene that is repaired imperfectly ata site that does not affect the coding sequence of the GA oxidase genein a manner that would eliminate the function of the encoded GA oxidaseprotein. For example, the site of the DSB or nick within the endogenouslocus may be in the upstream or 5′ region of the GA oxidase gene (e.g.,a promoter and/or enhancer sequence) to affect or reduce its level ofexpression. Similarly, such targeted knock-out or knock-down mutationsof a GA oxidase gene may be generated with a donor template molecule todirect a particular or desired mutation at or near the target site viarepair of the DSB or nick. The donor template molecule may comprise ahomologous sequence with or without an insertion sequence and comprisingone or more mutations, such as one or more deletions, insertions,inversions and/or substitutions, relative to the targeted genomicsequence at or near the site of the DSB or nick. For example, targetedknock-out mutations of a GA oxidase gene may be achieved by deleting orinverting at least a portion of the gene or by introducing a frame shiftor premature stop codon into the coding sequence of the gene. A deletionof a portion of a GA oxidase gene may also be introduced by generatingDSBs or nicks at two target sites and causing a deletion of theintervening target region flanked by the target sites.

A site-specific nuclease provided herein may be selected from the groupconsisting of a zinc-finger nuclease (ZFN), a meganuclease, anRNA-guided endonuclease, a TALE-endonuclease (TALEN), a recombinase, atransposase, or any combination thereof. See, e.g., Khandagale, K. etal., “Genome editing for targeted improvement in plants,” PlantBiotechnol Rep 10: 327-343 (2016); and Gaj, T. et al., “ZFN, TALEN andCRISPR/Cas-based methods for genome engineering,” Trends Biotechnol.31(7): 397-405 (2013), the contents and disclosures of which areincorporated herein by reference. A recombinase may be a serinerecombinase attached to a DNA recognition motif, a tyrosine recombinaseattached to a DNA recognition motif or other recombinase enzyme known inthe art. A recombinase or transposase may be a DNA transposase orrecombinase attached to a DNA binding domain. A tyrosine recombinaseattached to a DNA recognition motif may be selected from the groupconsisting of a Cre recombinase, a Flp recombinase, and a Tnp1recombinase. According to some embodiments, a Cre recombinase or a Ginrecombinase provided herein is tethered to a zinc-finger DNA bindingdomain. In another embodiment, a serine recombinase attached to a DNArecognition motif provided herein is selected from the group consistingof a PhiC31 integrase, an R4 integrase, and a TP-901 integrase. Inanother embodiment, a DNA transposase attached to a DNA binding domainprovided herein is selected from the group consisting of a TALE-piggyBacand TALE-Mutator.

According to embodiments of the present disclosure, an RNA-guidedendonuclease may be selected from the group consisting of Cas1, Cas1B,Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 andCsx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2,Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2,Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2,Csf3, Csf4, Cpf1, CasX, CasY, and homologs or modified versions thereof,Argonaute (non-limiting examples of Argonaute proteins include Thermusthermophilus Argonaute (TtAgo), Pyrococcus furiosus Argonaute (PfAgo),Natronobacterium gregoryi Argonaute (NgAgo) and homologs or modifiedversions thereof. According to some embodiments, an RNA-guidedendonuclease may be a Cas9 or Cpf1 enzyme.

In an aspect, a site-specific nuclease provided herein is selected fromthe group consisting of a zinc-finger nuclease, a meganuclease, anRNA-guided nuclease, a TALE-nuclease, a recombinase, a transposase, orany combination thereof. In another aspect, a site-specific nucleaseprovided herein is selected from the group consisting of a Cas9 or aCpf1. In another aspect, a site-specific nuclease provided herein isselected from the group consisting of a Cas1, a Cas1B, a Cas2, a Cas3, aCas4, a Cas5, a Cas6, a Cas7, a Cas8, a Cas9, a Cas10, a Csy1, a Csy2, aCsy3, a Cse1, a Cse2, a Csc1, a Csc2, a Csa5, a Csn2, a Csm2, a Csm3, aCsm4, a Csm5, a Csm6, a Cmr1, a Cmr3, a Cmr4, a Cmr5, a Cmr6, a Csb1, aCsb2, a Csb3, a Csx17, a Csx14, a Csx10, a Csx16, a CsaX, a Csx3, aCsx1, a Csx15, a Csf1, a Csf2, a Csf3, a Csf4, a Cpf1, CasX, CasY, ahomolog thereof, or a modified version thereof. In another aspect, anRNA-guided nuclease provided herein is selected from the groupconsisting of a Cas9 or a Cpf1. In another aspect, an RNA guidednuclease provided herein is selected from the group consisting of aCas1, a Cas1B, a Cas2, a Cas3, a Cas4, a Cas5, a Cas6, a Cas7, a Cas8, aCas9, a Cas10, a Csy1, a Csy2, a Csy3, a Cse1, a Cse2, a Csc1, a Csc2, aCsa5, a Csn2, a Csm2, a Csm3, a Csm4, a Csm5, a Csm6, a Cmr1, a Cmr3, aCmr4, a Cmr5, a Cmr6, a Csb1, a Csb2, a Csb3, a Csx17, a Csx14, a Csx10,a Csx16, a CsaX, a Csx3, a Csx1, a Csx15, a Csf1, a Csf2, a Csf3, aCsf4, a Cpf1, CasX, CasY, a homolog thereof, or a modified versionthereof. In another aspect, a method and/or a composition providedherein comprises at least one, at least two, at least three, at leastfour, at least five, at least six, at least seven, at least eight, atleast nine, or at least ten site-specific nucleases. In yet anotheraspect, a method and/or a composition provided herein comprises at leastone, at least two, at least three, at least four, at least five, atleast six, at least seven, at least eight, at least nine, or at leastten polynucleotides encoding at least one, at least two, at least three,at least four, at least five, at least six, at least seven, at leasteight, at least nine, or at least ten site-specific nucleases.

For RNA-guided endonucleases, a guide RNA (gRNA) molecule is furtherprovided to direct the endonuclease to a target site in the genome ofthe plant via base-pairing or hybridization to cause a DSB or nick at ornear the target site. The gRNA may be transformed or introduced into aplant cell or tissue (perhaps along with a nuclease, ornuclease-encoding DNA molecule, construct or vector) as a gRNA molecule,or as a recombinant DNA molecule, construct or vector comprising atranscribable DNA sequence encoding the guide RNA operably linked to aplant-expressible promoter. As understood in the art, a “guide RNA” maycomprise, for example, a CRISPR RNA (crRNA), a single-chain guide RNA(sgRNA), or any other RNA molecule that may guide or direct anendonuclease to a specific target site in the genome. A “single-chainguide RNA” (or “sgRNA”) is a RNA molecule comprising a crRNA covalentlylinked a tracrRNA by a linker sequence, which may be expressed as asingle RNA transcript or molecule. The guide RNA comprises a guide ortargeting sequence that is identical or complementary to a target sitewithin the plant genome, such as at or near a GA oxidase gene. Aprotospacer-adjacent motif (PAM) may be present in the genomeimmediately adjacent and upstream to the 5′ end of the genomic targetsite sequence complementary to the targeting sequence of the guideRNA—i.e., immediately downstream (3′) to the sense (+) strand of thegenomic target site (relative to the targeting sequence of the guideRNA) as known in the art. See, e.g., Wu, X. et al., “Target specificityof the CRISPR-Cas9 system,” Quant Biol. 2(2): 59-70 (2014), the contentand disclosure of which is incorporated herein by reference. The genomicPAM sequence on the sense (+) strand adjacent to the target site(relative to the targeting sequence of the guide RNA) may comprise5′-NGG-3′. However, the corresponding sequence of the guide RNA (i.e.,immediately downstream (3′) to the targeting sequence of the guide RNA)may generally not be complementary to the genomic PAM sequence. Theguide RNA may typically be a non-coding RNA molecule that does notencode a protein. The guide sequence of the guide RNA may be at least 10nucleotides in length, such as 12-40 nucleotides, 12-30 nucleotides,12-20 nucleotides, 12-35 nucleotides, 12-30 nucleotides, 15-30nucleotides, 17-30 nucleotides, or 17-25 nucleotides in length, or about12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or morenucleotides in length. The guide sequence may be at least 95%, at least96%, at least 97%, at least 99% or 100% identical or complementary to atleast 10, at least 11, at least 12, at least 13, at least 14, at least15, at least 16, at least 17, at least 18, at least 19, at least 20, atleast 21, at least 22, at least 23, at least 24, at least 25, or moreconsecutive nucleotides of a DNA sequence at the genomic target site.

For genome editing at or near the GA20 oxidase_3 gene with an RNA-guidedendonuclease, a guide RNA may be used comprising a guide sequence thatis at least 90%, at least 95%, at least 96%, at least 97%, at least 99%or 100% identical or complementary to at least 10, at least 11, at least12, at least 13, at least 14, at least 15, at least 16, at least 17, atleast 18, at least 19, at least 20, at least 21, at least 22, at least23, at least 24, at least 25, or more consecutive nucleotides of SEQ IDNO: 34 or a sequence complementary thereto (e.g., 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25 or more consecutive nucleotides ofSEQ ID NO: 34 or a sequence complementary thereto). For genome editingat or near the GA20 oxidase_5 gene with an RNA-guided endonuclease, aguide RNA may be used comprising a guide sequence that is at least 90%,at least 95%, at least 96%, at least 97%, at least 99% or 100% identicalor complementary to at least 10, at least 11, at least 12, at least 13,at least 14, at least 15, at least 16, at least 17, at least 18, atleast 19, at least 20, at least 21, at least 22, at least 23, at least24, at least 25, or more consecutive nucleotides of SEQ ID NO: 35 or asequence complementary thereto (e.g., 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25 or more consecutive nucleotides of SEQ IDNO: 35 or a sequence complementary thereto). As used herein, the term“consecutive” in reference to a polynucleotide or protein sequence meanswithout deletions or gaps in the sequence.

For knockdown (and possibly knockout) mutations through genome editing,an RNA-guided endonuclease may be targeted to an upstream or downstreamsequence, such as a promoter and/or enhancer sequence, or an intron,5′UTR, and/or 3′UTR sequence of a GA20 oxidase_3 or GA20 oxidase_5 geneto mutate one or more promoter and/or regulatory sequences of the geneand affect or reduce its level of expression. For knockdown (andpossibly knockout) of the GA20 oxidase_3 gene in corn, a guide RNA maybe used comprising a guide sequence that is at least 90%, at least 95%,at least 96%, at least 97%, at least 99% or 100% identical orcomplementary to at least 10, at least 11, at least 12, at least 13, atleast 14, at least 15, at least 16, at least 17, at least 18, at least19, at least 20, at least 21, at least 22, at least 23, at least 24, atleast 25, or more consecutive nucleotides within the nucleotide sequencerange 1-3096 of SEQ ID NO: 34, the nucleotide sequence range 3666-3775of SEQ ID NO: 34, the nucleotide sequence range 4098-5314 of SEQ ID NO:34, the nucleotide sequence range 5585-5800 of SEQ ID NO: 34, or thenucleotide sequence range 5801-8800 of SEQ ID NO: 34, or a sequencecomplementary thereto (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25 or more consecutive nucleotides within the nucleotidesequence range 1-3096, 3666-3775, 4098-5314, 5585-5800, 5801-8800, or5585-8800 of SEQ ID NO: 34, or a sequence complementary thereto).

For knockdown (and possibly knockout) of the GA20 oxidase_5 gene incorn, a guide RNA may be used comprising a guide sequence that is atleast 90%, at least 95%, at least 96%, at least 97%, at least 99% or100% identical or complementary to at least 10, at least 11, at least12, at least 13, at least 14, at least 15, at least 16, at least 17, atleast 18, at least 19, at least 20, at least 21, at least 22, at least23, at least 24, at least 25, or more consecutive nucleotides within thenucleotide sequence range 1-3000 of SEQ ID NO: 35, the nucleotidesequence range 1-3000 of SEQ ID NO: 35, the nucleotide sequence range3792-3906 of SEQ ID NO: 35, the nucleotide sequence range 4476-5197 ofSEQ ID NO: 35, or the nucleotide sequence range 5860-8859 of SEQ ID NO:35, or a sequence complementary thereto (e.g., 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more consecutive nucleotideswithin the nucleotide sequence range 1-3000, 3792-3906, 4476-5197, or5860-8859 of SEQ ID NO: 35, or a sequence complementary thereto).

For knockout (and possibly knockdown) mutations through genome editing,an RNA-guided endonuclease may be targeted to a coding and/or intronsequence of a GA20 oxidase_3 or GA20 oxidase_5 gene to potentiallyeliminate expression and/or activity of a functional GA oxidase proteinfrom the gene. However, a knockout of a GA oxidase gene expression mayalso be achieved in some cases by targeting the upstream and/or 5′UTRsequence(s) of the gene, or other sequences at or near the genomic locusof the gene. Thus, a knockout of a GA oxidase gene expression may beachieved by targeting a genomic sequence at or near the site or locus ofa targeted GA20 oxidase_3 or GA20 oxidase_5 gene, an upstream ordownstream sequence, such as a promoter and/or enhancer sequence, or anintron, 5′UTR, and/or 3′UTR sequence, of a GA20 oxidase_3 or GA20oxidase_5 gene, as described above for knockdown of a GA20 oxidase_3 orGA20 oxidase_5 gene.

For knockout (and possibly knockdown) of the GA20 oxidase_3 gene incorn, a guide RNA may be used comprising a guide sequence that is atleast 90%, at least 95%, at least 96%, at least 97%, at least 99% or100% identical or complementary to at least 10, at least 11, at least12, at least 13, at least 14, at least 15, at least 16, at least 17, atleast 18, at least 19, at least 20, at least 21, at least 22, at least23, at least 24, at least 25, or more consecutive nucleotides within thenucleotide sequence range 3097-5584 of SEQ ID NO: 34, the nucleotidesequence range 3097-3665 of SEQ ID NO: 34, the nucleotide sequence range3776-4097 of SEQ ID NO: 34, or the nucleotide sequence range 5315-5584of SEQ ID NO: 34, or a sequence complementary thereto (e.g., 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more consecutivenucleotides within the nucleotide sequence range 3097-5584, 3097-3665,3097-3775, 3665-4097, 3776-4097, 3776-5314, 4098-5584, or 5315-5584 ofSEQ ID NO: 34, or a sequence complementary thereto).

For knockout (and possibly knockdown) of the GA20 oxidase_5 gene incorn, a guide RNA may be used comprising a guide sequence that is atleast 90%, at least 95%, at least 96%, at least 97%, at least 99% or100% identical or complementary to at least 10, at least 11, at least12, at least 13, at least 14, at least 15, at least 16, at least 17, atleast 18, at least 19, at least 20, at least 21, at least 22, at least23, at least 24, at least 25, or more consecutive nucleotides within thenucleotide sequence range 3001-5473 of SEQ ID NO: 35, the nucleotidesequence range 3001-3791 of SEQ ID NO: 35, the nucleotide sequence range3907-4475 of SEQ ID NO: 35, or the nucleotide sequence range 5198-5473of SEQ ID NO: 35, or a sequence complementary thereto (e.g., 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more consecutivenucleotides within the nucleotide sequence range 3001-5473, 3001-3791,3001-3906, 3792-4475, 3907-4475, 3907-5197, 4476-5473, or 5198-5473 ofSEQ ID NO: 35, or a sequence complementary thereto).

According to some embodiments, a guide RNA for targeting an endogenousGA20 oxidase_3 and/or GA20 oxidase_5 gene is provided comprising a guidesequence that is at least 90%, at least 95%, at least 96%, at least 97%,at least 99% or 100% identical or complementary to at least 10, at least11, at least 12, at least 13, at least 14, at least 15, at least 16, atleast 17, at least 18, at least 19, at least 20, or at least 21consecutive nucleotides of any one or more of SEQ ID NOs: 138-167.

For genome editing at or near the GA20 oxidase_4 gene with an RNA-guidedendonuclease, a guide RNA may be used comprising a guide sequence thatis at least 90%, at least 95%, at least 96%, at least 97%, at least 99%or 100% identical or complementary to at least 10, at least 11, at least12, at least 13, at least 14, at least 15, at least 16, at least 17, atleast 18, at least 19, at least 20, at least 21, at least 22, at least23, at least 24, at least 25, or more consecutive nucleotides of SEQ IDNO: 38 or a sequence complementary thereto (e.g., 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25 or more consecutive nucleotides ofSEQ ID NO: 38 or a sequence complementary thereto).

For knockout (and possibly knockdown) mutations through genome editing,an RNA-guided endonuclease may be targeted to a coding and/or intronsequence of a GA20 oxidase_4 gene to potentially eliminate expressionand/or activity of a functional GA20 oxidase_4 protein from the gene.For the GA20 oxidase_4 gene in corn, a guide RNA may be used comprisinga guide sequence that is at least 90%, at least 95%, at least 96%, atleast 97%, at least 99% or 100% identical or complementary to at least10, at least 11, at least 12, at least 13, at least 14, at least 15, atleast 16, at least 17, at least 18, at least 19, at least 20, at least21, at least 22, at least 23, at least 24, at least 25, or moreconsecutive nucleotides within the nucleotide sequence range 1544-2852of SEQ ID NO: 38, the nucleotide sequence range 1544-1995 of SEQ ID NO:38, the nucleotide sequence range 2084-2411 of SEQ ID NO: 38, or thenucleotide sequence range 2517-2852 of SEQ ID NO: 38, or a sequencecomplementary thereto (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25 or more consecutive nucleotides within the nucleotidesequence range 1544-2852, 1544-1995, 1544-2083, 1996-2411, 2084-2411,2084-2516, 2412-2852, or 2517-2852 of SEQ ID NO: 38, or a sequencecomplementary thereto).

For knockdown (and possibly knockout) mutations through genome editing,an RNA-guided endonuclease may be targeted to an upstream or downstreamsequence, such as a promoter and/or enhancer sequence, or an intron,5′UTR, and/or 3′UTR sequence of a GA20 oxidase_4 gene to mutate one ormore promoter and/or regulatory sequences of the gene and affect orreduce its level of expression. For knockdown of the GA20 oxidase_3 genein corn, a guide RNA may be used comprising a guide sequence that is atleast 90%, at least 95%, at least 96%, at least 97%, at least 99% or100% identical or complementary to at least 10, at least 11, at least12, at least 13, at least 14, at least 15, at least 16, at least 17, atleast 18, at least 19, at least 20, at least 21, at least 22, at least23, at least 24, at least 25, or more consecutive nucleotides within thenucleotide sequence range 1-1416 of SEQ ID NO: 38, the nucleotidesequence range 1417-1543 of SEQ ID NO: 38, the nucleotide sequence range1996-2083 of SEQ ID NO: 38, the nucleotide sequence range 2412-2516 ofSEQ ID NO: 38, the nucleotide sequence range 2853-3066 of SEQ ID NO: 38,or the nucleotide sequence range 3067-4465 of SEQ ID NO: 38, or asequence complementary thereto (e.g., 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25 or more consecutive nucleotides withinthe nucleotide sequence range 1-1416, 1417-1543, 1-1543, 1996-2083,2412-2516, 2853-3066, 3067-4465 or 2853-4465 of SEQ ID NO: 38, or asequence complementary thereto).

In addition to the guide sequence, a guide RNA may further comprise oneor more other structural or scaffold sequence(s), which may bind orinteract with an RNA-guided endonuclease. Such scaffold or structuralsequences may further interact with other RNA molecules (e.g.,tracrRNA). Methods and techniques for designing targeting constructs andguide RNAs for genome editing and site-directed integration at a targetsite within the genome of a plant using an RNA-guided endonuclease areknown in the art.

According to some embodiments, recombinant DNA constructs and vectorsare provided comprising a polynucleotide sequence encoding asite-specific nuclease, such as a zinc-finger nuclease (ZFN), ameganuclease, an RNA-guided endonuclease, a TALE-endonuclease (TALEN), arecombinase, or a transposase, wherein the coding sequence is operablylinked to a plant expressible promoter. For RNA-guided endonucleases,recombinant DNA constructs and vectors are further provided comprising apolynucleotide sequence encoding a guide RNA, wherein the guide RNAcomprises a guide sequence of sufficient length having a percentidentity or complementarity to a target site within the genome of aplant, such as at or near a targeted GA oxidase gene. According to someembodiments, a polynucleotide sequence of a recombinant DNA constructand vector that encodes a site-specific nuclease or a guide RNA may beoperably linked to a plant expressible promoter, such as an induciblepromoter, a constitutive promoter, a tissue-specific promoter, etc.

According to some embodiments, a recombinant DNA construct or vector maycomprise a first polynucleotide sequence encoding a site-specificnuclease and a second polynucleotide sequence encoding a guide RNA thatmay be introduced into a plant cell together via plant transformationtechniques. Alternatively, two recombinant DNA constructs or vectors maybe provided including a first recombinant DNA construct or vector and asecond DNA construct or vector that may be introduced into a plant celltogether or sequentially via plant transformation techniques, whereinthe first recombinant DNA construct or vector comprises a polynucleotidesequence encoding a site-specific nuclease and the second recombinantDNA construct or vector comprises a polynucleotide sequence encoding aguide RNA. According to some embodiments, a recombinant DNA construct orvector comprising a polynucleotide sequence encoding a site-specificnuclease may be introduced via plant transformation techniques into aplant cell that already comprises (or is transformed with) a recombinantDNA construct or vector comprising a polynucleotide sequence encoding aguide RNA. Alternatively, a recombinant DNA construct or vectorcomprising a polynucleotide sequence encoding a guide RNA may beintroduced via plant transformation techniques into a plant cell thatalready comprises (or is transformed with) a recombinant DNA constructor vector comprising a polynucleotide sequence encoding a site-specificnuclease. According to yet further embodiments, a first plant comprising(or transformed with) a recombinant DNA construct or vector comprising apolynucleotide sequence encoding a site-specific nuclease may be crossedwith a second plant comprising (or transformed with) a recombinant DNAconstruct or vector comprising a polynucleotide sequence encoding aguide RNA. Such recombinant DNA constructs or vectors may be transientlytransformed into a plant cell or stably transformed or integrated intothe genome of a plant cell.

In an aspect, vectors comprising polynucleotides encoding asite-specific nuclease, and optionally one or more, two or more, threeor more, or four or more gRNAs are provided to a plant cell bytransformation methods known in the art (e.g., without being limiting,particle bombardment, PEG-mediated protoplast transfection orAgrobacterium-mediated transformation). In an aspect, vectors comprisingpolynucleotides encoding a Cas9 nuclease, and optionally one or more,two or more, three or more, or four or more gRNAs are provided to aplant cell by transformation methods known in the art (e.g., withoutbeing limiting, particle bombardment, PEG-mediated protoplasttransfection or Agrobacterium-mediated transformation). In anotheraspect, vectors comprising polynucleotides encoding a Cpf1 and,optionally one or more, two or more, three or more, or four or morecrRNAs are provided to a cell by transformation methods known in the art(e.g., without being limiting, viral transfection, particle bombardment,PEG-mediated protoplast transfection or Agrobacterium-mediatedtransformation).

Several site-specific nucleases, such as recombinases, zinc fingernucleases (ZFNs), meganucleases, and TALENs, are not RNA-guided andinstead rely on their protein structure to determine their target sitefor causing the DSB or nick, or they are fused, tethered or attached toa DNA-binding protein domain or motif. The protein structure of thesite-specific nuclease (or the fused/attached/tethered DNA bindingdomain) may target the site-specific nuclease to the target site.According to many of these embodiments, non-RNA-guided site-specificnucleases, such as recombinases, zinc finger nucleases (ZFNs),meganucleases, and TALENs, may be designed, engineered and constructedaccording to known methods to target and bind to a target site at ornear the genomic locus of an endogenous GA oxidase gene of a corn orcereal plant, such as the GA20 oxidase_3 gene or the GA20 oxidase_5 genein corn, to create a DSB or nick at such genomic locus to knockout orknockdown expression of the GA oxidase gene via repair of the DSB ornick. For example, an engineered site-specific nuclease, such as arecombinase, zinc finger nuclease (ZFN), meganuclease, or TALEN, may bedesigned to target and bind to (i) a target site within the genome of aplant corresponding to a sequence within SEQ ID NO: 34, or itscomplementary sequence, to create a DSB or nick at the genomic locus forthe GA20 oxidase_3 gene, (ii) a target site within the genome of a plantcorresponding to a sequence within SEQ ID NO: 35, or its complementarysequence, to create a DSB or nick at the genomic locus for the GA20oxidase_5 gene, or (iii) a target site within the genome of a plantcorresponding to a sequence within SEQ ID NO: 38, or its complementarysequence, to create a DSB or nick at the genomic locus for the GA20oxidase_4 gene, which may then lead to the creation of a mutation orinsertion of a sequence at the site of the DSB or nick, through cellularrepair mechanisms, which may be guided by a donor molecule or template.

In an aspect, a targeted genome editing technique described herein maycomprise the use of a recombinase. In some embodiments, a tyrosinerecombinase attached, etc., to a DNA recognition domain or motif may beselected from the group consisting of a Cre recombinase, a Flprecombinase, and a Tnp1 recombinase. In an aspect, a Cre recombinase ora Gin recombinase provided herein may be tethered to a zinc-finger DNAbinding domain. The Flp-FRT site-directed recombination system may comefrom the 2μ plasmid from the baker's yeast Saccharomyces cerevisiae. Inthis system, Flp recombinase (flippase) may recombine sequences betweenflippase recognition target (FRT) sites. FRT sites comprise 34nucleotides. Flp may bind to the “arms” of the FRT sites (one arm is inreverse orientation) and cleaves the FRT site at either end of anintervening nucleic acid sequence. After cleavage, Flp may recombinenucleic acid sequences between two FRT sites. Cre-lox is a site-directedrecombination system derived from the bacteriophage P1 that is similarto the Flp-FRT recombination system. Cre-lox can be used to invert anucleic acid sequence, delete a nucleic acid sequence, or translocate anucleic acid sequence. In this system, Cre recombinase may recombine apair of lox nucleic acid sequences. Lox sites comprise 34 nucleotides,with the first and last 13 nucleotides (arms) being palindromic. Duringrecombination, Cre recombinase protein binds to two lox sites ondifferent nucleic acids and cleaves at the lox sites. The cleavednucleic acids are spliced together (reciprocally translocated) andrecombination is complete. In another aspect, a lox site provided hereinis a loxP, lox 2272, loxN, lox 511, lox 5171, lox71, lox66, M2, M3, M7,or M11 site.

ZFNs are synthetic proteins consisting of an engineered zinc fingerDNA-binding domain fused to a cleavage domain (or a cleavagehalf-domain), which may be derived from a restriction endonuclease(e.g., FokI). The DNA binding domain may be canonical (C2H2) ornon-canonical (e.g., C3H or C4). The DNA-binding domain can comprise oneor more zinc fingers (e.g., 2, 3, 4, 5, 6, 7, 8, 9 or more zinc fingers)depending on the target site. Multiple zinc fingers in a DNA-bindingdomain may be separated by linker sequence(s). ZFNs can be designed tocleave almost any stretch of double-stranded DNA by modification of thezinc finger DNA-binding domain. ZFNs form dimers from monomers composedof a non-specific DNA cleavage domain (e.g., derived from the FokInuclease) fused to a DNA-binding domain comprising a zinc finger arrayengineered to bind a target site DNA sequence. The DNA-binding domain ofa ZFN may typically be composed of 3-4 (or more) zinc-fingers. The aminoacids at positions −1, +2, +3, and +6 relative to the start of the zincfinger a-helix, which contribute to site-specific binding to the targetsite, can be changed and customized to fit specific target sequences.The other amino acids may form a consensus backbone to generate ZFNswith different sequence specificities. Methods and rules for designingZFNs for targeting and binding to specific target sequences are known inthe art. See, e.g., US Patent App. Nos. 2005/0064474, 2009/0117617, and2012/0142062, the contents and disclosures of which are incorporatedherein by reference. The FokI nuclease domain may require dimerizationto cleave DNA and therefore two ZFNs with their C-terminal regions areneeded to bind opposite DNA strands of the cleavage site (separated by5-7 bp). The ZFN monomer can cut the target site if the two-ZF-bindingsites are palindromic. A ZFN, as used herein, is broad and includes amonomeric ZFN that can cleave double stranded DNA without assistancefrom another ZFN. The term ZFN may also be used to refer to one or bothmembers of a pair of ZFNs that are engineered to work together to cleaveDNA at the same site.

Without being limited by any scientific theory, because the DNA-bindingspecificities of zinc finger domains can be re-engineered using one ofvarious methods, customized ZFNs can theoretically be constructed totarget nearly any target sequence (e.g., at or near a GA oxidase gene ina plant genome). Publicly available methods for engineering zinc fingerdomains include Context-dependent Assembly (CoDA), Oligomerized PoolEngineering (OPEN), and Modular Assembly. In an aspect, a method and/orcomposition provided herein comprises one or more, two or more, three ormore, four or more, or five or more ZFNs. In another aspect, a ZFNprovided herein is capable of generating a targeted DSB or nick. In anaspect, vectors comprising polynucleotides encoding one or more, two ormore, three or more, four or more, or five or more ZFNs are provided toa cell by transformation methods known in the art (e.g., without beinglimiting, viral transfection, particle bombardment, PEG-mediatedprotoplast transfection, or Agrobacterium-mediated transformation). TheZFNs may be introduced as ZFN proteins, as polynucleotides encoding ZFNproteins, and/or as combinations of proteins and protein-encodingpolynucleotides.

Meganucleases, which are commonly identified in microbes, such as theLAGLIDADG family of homing endonucleases, are unique enzymes with highactivity and long recognition sequences (>14 bp) resulting insite-specific digestion of target DNA. Engineered versions of naturallyoccurring meganucleases typically have extended DNA recognitionsequences (for example, 14 to 40 bp). According to some embodiments, ameganuclease may comprise a scaffold or base enzyme selected from thegroup consisting of I-CreI, I-CeuI, I-MsoI, I-SceI, I-AniI, and I-DmoI.The engineering of meganucleases can be more challenging than ZFNs andTALENs because the DNA recognition and cleavage functions ofmeganucleases are intertwined in a single domain. Specialized methods ofmutagenesis and high-throughput screening have been used to create novelmeganuclease variants that recognize unique sequences and possessimproved nuclease activity. Thus, a meganuclease may be selected orengineered to bind to a genomic target sequence in a plant, such as ator near the genomic locus of a GA oxidase gene. In an aspect, a methodand/or composition provided herein comprises one or more, two or more,three or more, four or more, or five or more meganucleases. In anotheraspect, a meganuclease provided herein is capable of generating atargeted DSB. In an aspect, vectors comprising polynucleotides encodingone or more, two or more, three or more, four or more, or five or moremeganucleases are provided to a cell by transformation methods known inthe art (e.g., without being limiting, viral transfection, particlebombardment, PEG-mediated protoplast transfection orAgrobacterium-mediated transformation).

TALENs are artificial restriction enzymes generated by fusing thetranscription activator-like effector (TALE) DNA binding domain to anuclease domain (e.g., Fold). When each member of a TALEN pair binds tothe DNA sites flanking a target site, the FokI monomers dimerize andcause a double-stranded DNA break at the target site. Besides thewild-type FokI cleavage domain, variants of the FokI cleavage domainwith mutations have been designed to improve cleavage specificity andcleavage activity. The FokI domain functions as a dimer, requiring twoconstructs with unique DNA binding domains for sites in the targetgenome with proper orientation and spacing. Both the number of aminoacid residues between the TALEN DNA binding domain and the FokI cleavagedomain and the number of bases between the two individual TALEN bindingsites are parameters for achieving high levels of activity.

TALENs are artificial restriction enzymes generated by fusing thetranscription activator-like effector (TALE) DNA binding domain to anuclease domain. In some aspects, the nuclease is selected from a groupconsisting of PvuII, MutH, TevI, FokI, AlwI, MlyI, SbfI, SdaI, StsI,CleDORF, Clo051, and Pept071. When each member of a TALEN pair binds tothe DNA sites flanking a target site, the FokI monomers dimerize andcause a double-stranded DNA break at the target site. The term TALEN, asused herein, is broad and includes a monomeric TALEN that can cleavedouble stranded DNA without assistance from another TALEN. The termTALEN is also refers to one or both members of a pair of TALENs thatwork together to cleave DNA at the same site.

Transcription activator-like effectors (TALEs) can be engineered to bindpractically any DNA sequence, such as at or near the genomic locus of aGA oxidase gene in a plant. TALE has a central DNA-binding domaincomposed of 13-28 repeat monomers of 33-34 amino acids. The amino acidsof each monomer are highly conserved, except for hypervariable aminoacid residues at positions 12 and 13. The two variable amino acids arecalled repeat-variable diresidues (RVDs). The amino acid pairs NI, NG,HD, and NN of RVDs preferentially recognize adenine, thymine, cytosine,and guanine/adenine, respectively, and modulation of RVDs can recognizeconsecutive DNA bases. This simple relationship between amino acidsequence and DNA recognition has allowed for the engineering of specificDNA binding domains by selecting a combination of repeat segmentscontaining the appropriate RVDs.

Besides the wild-type FokI cleavage domain, variants of the FokIcleavage domain with mutations have been designed to improve cleavagespecificity and cleavage activity. The FokI domain functions as a dimer,requiring two constructs with unique DNA binding domains for sites inthe target genome with proper orientation and spacing. Both the numberof amino acid residues between the TALEN DNA binding domain and the FokIcleavage domain and the number of bases between the two individual TALENbinding sites are parameters for achieving high levels of activity.PvuII, MutH, and TevI cleavage domains are useful alternatives to FokIand FokI variants for use with TALEs. PvuII functions as a highlyspecific cleavage domain when coupled to a TALE (see Yank et al. 2013.PLoS One. 8: e82539). MutH is capable of introducing strand-specificnicks in DNA (see Gabsalilow et al. 2013. Nucleic Acids Research. 41:e83). TevI introduces double-stranded breaks in DNA at targeted sites(see Beurdeley et al., 2013. Nature Communications. 4: 1762).

The relationship between amino acid sequence and DNA recognition of theTALE binding domain allows for designable proteins. Software programssuch as DNA Works can be used to design TALE constructs. Other methodsof designing TALE constructs are known to those of skill in the art. SeeDoyle et al., Nucleic Acids Research (2012) 40: W117-122.; Cermak etal., Nucleic Acids Research (2011). 39:e82; andtale-nt.cac.cornell.edu/about. In an aspect, a method and/or compositionprovided herein comprises one or more, two or more, three or more, fouror more, or five or more TALENs. In another aspect, a TALEN providedherein is capable of generating a targeted DSB. In an aspect, vectorscomprising polynucleotides encoding one or more, two or more, three ormore, four or more, or five or more TALENs are provided to a cell bytransformation methods known in the art (e.g., without being limiting,viral transfection, particle bombardment, PEG-mediated protoplasttransfection or Agrobacterium-mediated transformation). See, e.g., USPatent App. Nos. 2011/0145940, 2011/0301073, and 2013/0117869, thecontents and disclosures of which are incorporated herein by reference.

As used herein, a “targeted genome editing technique” refers to anymethod, protocol, or technique that allows the precise and/or targetedediting of a specific location in a genome of a plant (i.e., the editingis largely or completely non-random) using a site-specific nuclease,such as a meganuclease, a zinc-finger nuclease (ZFN), an RNA-guidedendonuclease (e.g., the CRISPR/Cas9 system), a TALE-endonuclease(TALEN), a recombinase, or a transposase. As used herein, “editing” or“genome editing” refers to generating a targeted mutation, deletion,inversion or substitution of at least 1, at least 2, at least 3, atleast 4, at least 5, at least 6, at least 7, at least 8, at least 9, atleast 10, at least 15, at least 20, at least 25, at least 30, at least35, at least 40, at least 45, at least 50, at least 75, at least 100, atleast 250, at least 500, at least 1000, at least 2500, at least 5000, atleast 10,000, or at least 25,000 nucleotides of an endogenous plantgenome nucleic acid sequence. As used herein, “editing” or “genomeediting” also encompasses the targeted insertion or site-directedintegration of at least 1, at least 2, at least 3, at least 4, at least5, at least 6, at least 7, at least 8, at least 9, at least 10, at least15, at least 20, at least 25, at least 30, at least 35, at least 40, atleast 45, at least 50, at least 75, at least 100, at least 250, at least500, at least 750, at least 1000, at least 1500, at least 2000, at least2500, at least 3000, at least 4000, at least 5000, at least 10,000, orat least 25,000 nucleotides into the endogenous genome of a plant. An“edit” or “genomic edit” in the singular refers to one such targetedmutation, deletion, inversion, substitution or insertion, whereas“edits” or “genomic edits” refers to two or more targeted mutation(s),deletion(s), inversion(s), substitution(s) and/or insertion(s), witheach “edit” being introduced via a targeted genome editing technique.

Given that suppression of GA20 oxidase_3, GA20 oxidase_4, and/or GA20oxidase_5 genes in corn produces plants having a shorter plant heightand internode length in addition to other beneficial traits, it isproposed that expression of one or more of these genes may be reduced oreliminated through genome editing one or more of these gene(s) toprovide similar beneficial traits to corn plants. Given further thatconstitutive expression of suppression constructs targeting these GA20oxidase genes produces corn plants having the beneficial short heighttraits without off-types in the ear, and that expression directly inreproductive ear tissues also does not give rise to reproductiveoff-types, it is proposed that one or more of these gene loci may beedited to knock-down or knock-out their expression to produce similareffects in corn plants. Targeted gene editing approaches could be usedto modify the sequence of the promoter and/or regulatory region(s) ofone or more of the GA20 oxidase_3, GA20 oxidase_4, and/or GA20 oxidase_5genes to knock-down or knock-out expression of these gene(s), such asthrough targeted deletions, insertions, mutations, or other sequencechanges. Indeed, the promoter and/or regulatory region(s) orsequence(s), or the 5′-UTR, 3′UTR, and/or intron sequence(s), of one ormore of the GA20 oxidase_3, GA20 oxidase_4, and/or GA20 oxidase_5 genesmay be largely deleted or mutated. Alternatively, all or a portion ofthe coding (exon), 5-UTR, 3′UTR, and/or intron sequence(s) of one ormore of the GA20 oxidase_3, GA20 oxidase_4, and/or GA20 oxidase_5 genesmay be edited, deleted, mutated, or otherwise modified to knock-down orknock-out expression or activity of these gene(s). Such targetedmodifications to the GA20 oxidase_3, GA20 oxidase_4, and/or GA20oxidase_5 gene loci may be achieved using any suitable genome editingtechnology known in the art, such as via repair of a double strand break(DSB) or nick introduced by a site-specific nuclease, such as, forexample, a zinc-finger nuclease, an engineered or native meganuclease, aTALE-endonuclease, or an RNA-guided endonuclease (e.g., Cas9 or Cpf1).Such repair of the DSB or nick may introduce spontaneous or stochasticdeletions, additions, mutations, etc., at the targeted site where theDSB or nick was introduced, or repair of the site may involve the use ofa donor template molecule to direct or cause a preferred or specificdeletion, addition, mutation, etc., at the targeted site.

As provided herein, a plant transformed with a recombinant DNA moleculeor transformation vector comprising a transgene encoding a transcribableDNA sequence encoding a non-coding RNA molecule that targets anendogenous GA oxidase gene for suppression may include a variety ofmonocot or cereal plants, such as maize/corn and other monocot or cerealplants that have separate male and female flowers (similarly to corn)and may thus be susceptible to off-types in female reproductive organs,structures or tissues with mutations to the GA pathway.

The present compositions and methods may be further applicable to othercereal plants that would benefit from a reduced plant height and/orincreased resistance to lodging. Such plants may be transformed withrecombinant DNA molecules or constructs to suppress one or moreendogenous GA20 and/or GA3 oxidase genes in the plant according to themethods and approaches provided herein to produce a cereal plant thatmay be shorter and/or resistant to lodging. Indeed, a cereal plantectopically expressing a transcribable DNA sequence encoding anon-coding RNA molecule that targets an endogenous GA oxidase gene forsuppression may have a variety of beneficial traits, such as shorterstature or plant height, shorter internode length, increased stalk/stemdiameter, improved lodging resistance, in addition to other improvedyield-related and/or drought tolerant traits as provided herein,relative to a wild-type or control plant not having the transgene ortranscribable DNA sequence. As described further below, cereal cropplants that have already been modified to have increased yield andresist lodging through mutations in the GA pathway, such as wheat, rice,millet, barley and sorghum, may instead be transformed with arecombinant DNA molecule or construct as provided herein. Unlike many ofthe GA pathway mutations in these crops which may be recessive,transgenic constructs expressing a suppression element targeting anendogenous biosynthetic GA oxidase gene in those crops may be dominanteven when hemizygous or present in the plant as a single copy. Thus,plants that may be transformed with a recombinant DNA molecule orconstruct expressing a suppression construct may potentially include avariety of monocot or cereal crops. Having a dominant transgenic locusthat causes a semi-dwarf, lodging resistant phenotype may beadvantageous and preferred over a recessive mutant allele for the samephenotype due to benefits in breeding and trait integration.

According to embodiments of the present disclosure, it is furtherproposed that GA oxidase genes in other cereal plants having thegreatest sequence identity/similarity to the GA20 oxidase_3, GA20oxidase_4, GA20 oxidase_5, GA3 oxidase_1, and/or GA3 oxidase_2 genes incorn that are shown herein to produce a short stature, semi-dwarfphenotype and other beneficial traits when suppressed with a recombinantDNA suppression construct, may also be targets for suppression toproduce transgenic cereal plants having similar semi-dwarf and/orlodging resistance phenotypes. Table 3 provides a list of GA oxidasegenes from other cereal plants (sorghum—Sorghum bicolor; rice—Oryzasativa; foxtail millet—Setaria Italica; wheat—Triticum aestivum; andbarley—Hordeum vulgare) having a high degree of sequence identity withone of the GA oxidase genes in corn that when suppressed produces ashort stature, semi-dwarf phenotype.

TABLE 3 Homologs of corn GA oxidase genes from other cereal crop plants.Cereal Corn cDNA CDS Protein Genomic Gene Name Species Homolog (SEQ IDNO) (SEQ ID NO) (SEQ ID NO) (SEQ ID NO) GA20 Sorghum GA20 Ox_3/ 84 85 8687 oxidase 2 bicolor GA20 Ox_5 GA20 Setaria GA20 Ox_3/ 88 89 90 91oxidase 2-like italica GA20 Ox_5 GA20 Oryza GA20 Ox_3/ 92 93 94 95oxidase 2 sativa GA20 Ox_5 GA20 Triticum GA20 Ox_3/ — 96 97 98oxidase-D2 aestivum GA20 Ox_5 Fe2OG Hordeum GA20 Ox_3/ 99 100 101 —dioxygenase vulgare GA20 Ox_5 Probable 2-ODD Sorghum GA20 Ox_4 102 103104 105 bicolor flavonol Setaria GA20 Ox_4 106 107 108 109synthase/flavanone italica 3-hydroxylase-like naringenin, 2- Oryza GA20Ox_4 110 111 112 113 oxoglutarate 3- sativa dioxygenase Fe2OG TriticumGA20 Ox_4 114 115 116 117 dioxygenase aestivum Fe2OG Hordeum GA20 Ox_4 —— 118 — dioxygenase vulgare GA3-beta- Sorghum GA3 Ox_1/ 119 120 121 122dioxygenase 2-2 bicolor GA3 Ox_2 GA3-beta- Setaria GA3 Ox_1/ 123 124 125126 dioxygenase 2-2- italica GA3 Ox_2 like GA3-beta- Oryza GA3 Ox_1/ 127128 129 130 dioxygenase 2-3 sativa GA3 Ox_2 GA3-beta- Hordeum GA3 Ox_1/131 132 133 — hydroxylase vulgare GA3 Ox_2 GA3ox- Triticum GA3 Ox_1/ 134135 136 137 D2 protein aestivum GA3 Ox_2

According to another aspect of the present disclosure, a recombinant DNAmolecule, vector or construct is provided for suppression of anendogenous GA oxidase (or GA oxidase-like) gene in a cereal plant, therecombinant DNA molecule, vector or construct comprising a transcribableDNA sequence encoding a non-coding RNA molecule, wherein the non-codingRNA molecule comprises a sequence that is (i) at least 80%, at least85%, at least 90%, at least 95%, at least 96%, at least 97%, at least98%, at least 99%, at least 99.5%, or 100% complementary to at least 15,at least 16, at least 17, at least 18, at least 19, at least 20, atleast 21, at least 22, at least 23, at least 24, at least 25, at least26, or at least 27 consecutive nucleotides of any one or more of SEQ IDNO: 84, 85, 87, 88, 89, 91, 92, 93, 95, 96, 98, 99, 100, 102, 103, 105,106, 107, 109, 110, 111, 113, 114, 115, 119, 120, 122, 123, 124, 126,127, 128, 130, 131, 132, 134, 135, and/or 137, and/or (ii) at least 80%,at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, at least 99.5%, or 100% complementary to atleast 15, at least 16, at least 17, at least 18, at least 19, at least20, at least 21, at least 22, at least 23, at least 24, at least 25, atleast 26, or at least 27 consecutive nucleotides of a mRNA moleculeencoding a protein in the cereal plant that is at least 80%, at least85%, at least 90%, at least 95%, at least 96%, at least 97%, at least98%, at least 99%, at least 99.5%, or 100% identical to any one or moreof SEQ ID NO: 86, 90, 94, 97, 101, 104, 108, 112, 116, 118, 121, 125,129, 133, and/or 136. Likewise, a non-coding RNA molecule may target anendogenous GA oxidase (or GA oxidase-like) gene in a cereal plant havinga percent identity to the GA oxidase gene(s) shown to affect plantheight in corn. Thus, a non-coding RNA molecule is further providedcomprising a sequence that is at least 80%, at least 85%, at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, atleast 99.5%, or 100% complementary to at least 15, at least 16, at least17, at least 18, at least 19, at least 20, at least 21, at least 22, atleast 23, at least 24, at least 25, at least 26, or at least 27consecutive nucleotides of a mRNA molecule encoding an endogenousprotein in a cereal plant that is at least 80%, at least 85%, at least90%, at least 95%, at least 96%, at least 97%, at least 98%, at least99%, at least 99.5%, or 100% identical to any one or more of SEQ ID NO:9, 12, 15, 30, and/or 33. As mentioned above, the non-coding RNAmolecule may target an exon, intron and/or UTR sequence of a GA oxidase(or GA oxidase-like) gene.

Further provided are methods for introducing or transforming into acereal plant, plant part, or plant cell any of the foregoing constructs,vectors, or constructs, according to any of the methods describedherein, which may be constructed in any suitable manner described hereinincluding different stacking or joint targeting arrangements, as well asmodified cereal plants, plant parts, plant tissues, and plant cells madethereby and/or comprising any such recombinant DNA molecule, vector orconstruct. Since a non-coding RNA molecule expressed from the aboveconstructs would be designed to target an endogenous GA oxidase gene,the cereal plant transformed with such recombinant DNA molecules,vectors or constructs should preferably correspond to the species oforigin for the target sequence, or closely related species, strains,germplasms, lines, etc. For example, a suppression constructcomplementary to SEQ ID NO: 84 should be used to transform a sorghumplant, such as a Sorghum bicolor plant, or perhaps related sorghumspecies, strains, etc., that would be expected to have a closely relatedor similar GA oxidase (or GA oxidase-like) gene sequence.

The genomic sequences for each of the above identified genes from cerealplants are further provided in Table 3, which may be used to targetthose genes for genome editing according to any known technique. Anysite-specific nuclease and method may be used as described herein togenerate a DSB or nick at or near the genomic locus for the gene, whichmay be repaired imperfectly or via template-mediated recombination tocreate mutations, etc., at, near or within the gene. Suitable nucleasesmay be selected from the group consisting of a zinc-finger nuclease(ZFN), a meganuclease, an RNA-guided endonuclease, a TALE-endonuclease(TALEN), a recombinase, a transposase, or any combination thereof. Foran RNA-guided endonuclease, a recombinant DNA construct or vector isprovided comprising a guide RNA may be used to direct the nuclease tothe target site. Accordingly, a guide RNA for editing a GA oxidase (orGA-oxidase-like) gene in a cereal crop may comprise a guide sequencethat is at least 90%, at least 95%, at least 96%, at least 97%, at least99% or 100% identical or complementary to at least 10, at least 11, atleast 12, at least 13, at least 14, at least 15, at least 16, at least17, at least 18, at least 19, at least 20, at least 21, at least 22, atleast 23, at least 24, at least 25, or more consecutive nucleotides ofany one or more of SEQ ID NO: 84, 85, 87, 88, 89, 91, 92, 93, 95, 96,98, 99, 100, 102, 103, 105, 106, 107, 109, 110, 111, 113, 114, 115, 119,120, 122, 123, 124, 126, 127, 128, 130, 131, 132, 134, 135, and/or 137.For site-specific nucleases that are not RNA-guided, such as azinc-finger nuclease (ZFN), a meganuclease, a TALE-endonuclease (TALEN),a recombinase, and/or a transposase, the genomic target specificity forediting is determined by its protein structure, particularly its DNAbinding domain. Such site-specific nucleases may be chosen, designed orengineered to bind and cut a desired target site at or near any of theabove GA oxidase (or GA oxidase-like) genes within the genome of acereal plant. Similar to transformation with a suppression construct, acereal plant transformed with a particular guide RNA, or a recombinantDNA molecule, vector or construct encoding a guide RNA, shouldpreferably be the species in which the targeted genomic sequence exists,or a closely related species, strain, germplasm, line, etc., such thatthe guide RNA is able to recognize and bind to the desired target cutsite.

Further provided are methods for introducing or transforming into acereal plant, plant part, or plant cell any guide RNA described above,or any construct, vector, or construct encoding such a guide RNA,perhaps in addition to an RNA-guided nuclease, according to any of themethods described herein, as well as modified cereal plants, plantparts, plant tissues, and plant cells made thereby and/or comprising anysuch recombinant DNA molecule, vector or construct and/or an edited GAoxidase (or GA oxidase-like) gene. Modified cereal plants having anedited GA oxidase (or GA oxidase-like) gene, and/or a suppressionelement targeting a GA oxidase (or GA oxidase-like) gene, may have oneor more beneficial traits provided herein, such as a shorter plantheight, shorter internode length, increased stalk/stem diameter,improved lodging resistance, and/or drought tolerance, relative to awild-type or control plant not having any such edit or suppressionelement. In addition to genome editing, mutations in a GA oxidase (or GAoxidase-like) gene may be introduced through other mutagenesistechniques as described herein

According to another aspect of the present disclosure, a transgenicplant(s), plant cell(s), seed(s), and plant part(s) are providedcomprising a transformation event or insertion into the genome of atleast one plant cell thereof, wherein the transformation event orinsertion comprises a recombinant DNA sequence, construct or expressioncassette comprising a transcribable DNA sequence encoding a non-codingRNA molecule that targets an endogenous GA oxidase gene for suppression,wherein the transcribable DNA sequence is operably linked to aplant-expressible promoter, such as a constitutive, vascular and/or leafpromoter. Such a transgenic plant may be produced by any suitabletransformation method as provided above, to produce a transgenic R₀plant, which may then be selfed or crossed to other plants to generateR₁ seed and subsequent progeny generations and seed through additionalcrosses, etc. Embodiments of the present disclosure further include aplant cell, tissue, explant, plant part, etc., comprising one or moretransgenic cells having a transformation event or genomic insertion of arecombinant DNA or polynucleotide sequence comprising a transcribableDNA sequence encoding a non-coding RNA molecule that targets anendogenous GA oxidase gene for suppression.

Transgenic plants, plant cells, seeds, and plant parts of the presentdisclosure may be homozygous or hemizygous for a transgenic event orinsertion of a transcribable DNA sequence for suppression of a GAoxidase gene into the genome of at least one plant cell thereof, or atargeted genome editing event, and plants, plant cells, seeds, and plantparts of the present embodiments may contain any number of copies ofsuch transgenic event(s), insertion(s) and/or edit(s). The dosage oramount of expression of a transgene or transcribable DNA sequence may bealtered by its zygosity and/or number of copies, which may affect thedegree or extent of phenotypic changes in the transgenic plant, etc. Asintroduced above, transgenic plants provided herein may include avariety of monocot or cereal plants, and even crop plants, such aswheat, rice and sorghum, already having increased yield and/or lodgingresistance due to prior breeding efforts and mutations of the GA pathwayin these plants. Advantages of using a transgene or transcribable DNAsequence to express a suppression element targeting a biosynthetic GAoxidase gene include not only the ability to limit expression in atissue-specific or tissue-preferred manner, but also the potentialdominance (e.g., dominant negative effects) of a single or hemizygouscopy of the transcribable DNA sequence to cause the beneficialshort-stature, semi-dwarf traits or phenotypes in crop plants. Thus,recombinant DNA molecules or constructs of the present disclosure may beused to create beneficial traits in a variety of monocot or cerealplants without off-types using only a single copy of the transgenicevent, insertion or construct. Unlike previously described mutations oralleles in the GA pathway that are recessive and require plants to behomozygous for the mutant allele, plants transformed with theGA-modifying transgenes and suppression constructs of the presentdisclosure may improve traits, yield and crop breeding efforts byfacilitating the production of hybrid cereal plants since they onlyrequire a single or hemizygous copy of the transgene or suppressionconstruct.

According to some embodiments, a transgenic or modified cereal or cornplant comprising a GA oxidase transgene or transcribable DNA sequencefor suppression of an endogenous GA oxidase gene, or a genome edited GAoxidase gene, may be further characterized as having one or morebeneficial traits, such as a shorter stature or semi-dwarf plant height,reduced internode length, increased stalk/stem diameter, improvedlodging resistance, reduced green snap, deeper roots, increased leafarea, earlier canopy closure, increased foliar water content and/orhigher stomatal conductance under water limiting conditions, reducedanthocyanin content and/or area in leaves under normal or nitrogen orwater limiting stress conditions, improved yield-related traitsincluding a larger female reproductive organ or ear, an increase in earweight, harvest index, yield, seed or kernel number, and/or seed orkernel weight, relative to a wild type or control plant. Such atransgenic cereal or corn plant may further have increased stresstolerance, such as increased drought tolerance, nitrogen utilization,and/or tolerance to high density planting.

For purposes of the present disclosure, a “plant” includes an explant,plant part, seedling, plantlet or whole plant at any stage ofregeneration or development. As used herein, a “transgenic plant” refersto a plant whose genome has been altered by the integration or insertionof a recombinant DNA molecule, construct or sequence. A transgenic plantincludes an R₀ plant developed or regenerated from an originallytransformed plant cell(s) as well as progeny transgenic plants in latergenerations or crosses from the R₀ transgenic plant. As used herein, a“plant part” may refer to any organ or intact tissue of a plant, such asa meristem, shoot organ/structure (e.g., leaf, stem or node), root,flower or floral organ/structure (e.g., bract, sepal, petal, stamen,carpel, anther and ovule), seed (e.g., embryo, endosperm, and seedcoat), fruit (e.g., the mature ovary), propagule, or other plant tissues(e.g., vascular tissue, dermal tissue, ground tissue, and the like), orany portion thereof. Plant parts of the present disclosure may beviable, nonviable, regenerable, and/or non-regenerable. A “propagule”may include any plant part that can grow into an entire plant.

According to present embodiments, a plant cell transformed with aconstruct or molecule comprising a transcribable DNA sequence forsuppression of an endogenous GA oxidase gene, or with a construct usedfor genome editing, may include any plant cell that is competent fortransformation as understood in the art based on the method oftransformation, such as a meristem cell, an embryonic cell, a calluscell, etc. As used herein, a “transgenic plant cell” simply refers toany plant cell that is transformed with a stably-integrated recombinantDNA molecule, construct or sequence. A transgenic plant cell may includean originally-transformed plant cell, a transgenic plant cell of aregenerated or developed R₀ plant, a transgenic plant cell cultured fromanother transgenic plant cell, or a transgenic plant cell from anyprogeny plant or offspring of the transformed R₀ plant, includingcell(s) of a plant seed or embryo, or a cultured plant cell, calluscell, etc.

Embodiments of the present disclosure further include methods for makingor producing transgenic or modified plants, such as by transformation,genome editing, crossing, etc., wherein the method comprises introducinga recombinant DNA molecule, construct or sequence comprising a GAoxidase transgene or a transcribable DNA sequence for suppression of anendogenous GA oxidase gene into a plant cell, or editing the genomiclocus of an endogenous GA oxidase gene, and then regenerating ordeveloping the transgenic or modified plant from the transformed oredited plant cell, which may be performed under selection pressurefavoring a transgenic event. Such methods may comprise transforming aplant cell with a recombinant DNA molecule, construct or sequencecomprising the transcribable DNA sequence for suppression of anendogenous GA oxidase gene, and selecting for a plant having one or morealtered phenotypes or traits, such as one or more of the followingtraits at one or more stages of development: shorter or semi-dwarfstature or plant height, shorter internode length in one or moreinternode(s), increased stalk/stem diameter, improved lodgingresistance, reduced green snap, deeper roots, increased leaf area,earlier canopy closure, increased foliar water content and/or higherstomatal conductance under water limiting conditions, reducedanthocyanin content and/or area in leaves under normal or nitrogen orwater limiting stress conditions, improved yield-related traitsincluding a larger female reproductive organ or ear, an increase in earweight, harvest index, yield, seed or kernel number, and/or seed orkernel weight, increased stress tolerance, such as increased droughttolerance, increased nitrogen utilization, and/or increased tolerance tohigh density planting, as compared to a wild type or control plant.

According to another aspect of the present disclosure, methods areprovided for planting a modified or transgenic plant(s) provided hereinat a normal/standard or high density in field. According to someembodiments, the yield of a crop plant per acre (or per land area) maybe increased by planting a modified or transgenic plant(s) of thepresent disclosure at a higher density in the field. As describedherein, modified or transgenic plants expressing a transcribable DNAsequence that encodes a non-coding RNA molecule targeting an endogenousGA oxidase gene for suppression, or having a genome-edited GA oxidasegene, may have reduced plant height, shorter internode(s), increasedstalk/stem diameter, and/or increased lodging resistance. It is proposedthat modified or transgenic plants may tolerate high density plantingconditions since an increase in stem diameter may resist lodging and theshorter plant height may allow for increased light penetrance to thelower leaves under high density planting conditions. Thus, modified ortransgenic plants provided herein may be planted at a higher density toincrease the yield per acre (or land area) in the field. For row crops,higher density may be achieved by planting a greater number ofseeds/plants per row length and/or by decreasing the spacing betweenrows.

According to some embodiments, a modified or transgenic crop plant maybe planted at a density in the field (plants per land/field area) thatis at least 5%, 10%, 15%, 20%, 25%, 50%, 75%, 100%, 125%, 150%, 175%,200%, 225%, or 250% higher than the normal planting density for thatcrop plant according to standard agronomic practices. A modified ortransgenic crop plant may be planted at a density in the field of atleast 38,000 plants per acre, at least 40,000 plants per acre, at least42,000 plants per acre, at least 44,000 plants per acre, at least 45,000plants per acre, at least 46,000 plants per acre, at least 48,000 plantsper acre, 50,000 plants per acre, at least 52,000 plants per acre, atleast 54,000 per acre, or at least 56,000 plants per acre. As anexample, corn plants may be planted at a higher density, such as in arange from about 38,000 plants per acre to about 60,000 plants per acre,or about 40,000 plants per acre to about 58,000 plants per acre, orabout 42,000 plants per acre to about 58,000 plants per acre, or about40,000 plants per acre to about 45,000 plants per acre, or about 45,000plants per acre to about 50,000 plants per acre, or about 50,000 plantsper acre to about 58,000 plants per acre, or about 52,000 plants peracre to about 56,000 plants per acre, or about 38,000 plants per acre,about 42,000 plant per acre, about 46,000 plant per acre, or about48,000 plants per acre, about 50,000 plants per acre, or about 52,000plants per acre, or about 54,000 plant per acre, as opposed to astandard density range, such as about 18,000 plants per acre to about38,000 plants per acre.

According to embodiments of the present disclosure, a modified cornplant(s) is/are provided that comprise (i) a plant height of less than2000 mm, less than 1950 mm, less than 1900 mm, less than 1850 mm, lessthan 1800 mm, less than 1750 mm, less than 1700 mm, less than 1650 mm,less than 1600 mm, less than 1550 mm, less than 1500 mm, less than 1450mm, less than 1400 mm, less than 1350 mm, less than 1300 mm, less than1250 mm, less than 1200 mm, less than 1150 mm, less than 1100 mm, lessthan 1050 mm, or less than 1000 mm, and/or (ii) an average stem or stalkdiameter of at least 18 mm, at least 18.5 mm, at least 19 mm, at least19.5 mm, at least 20 mm, at least 20.5 mm, at least 21 mm, at least 21.5mm, or at least 22 mm. Stated a different way, a modified corn plant(s)is/are provided that comprise a plant height of less than 2000 mm, lessthan 1950 mm, less than 1900 mm, less than 1850 mm, less than 1800 mm,less than 1750 mm, less than 1700 mm, less than 1650 mm, less than 1600mm, less than 1550 mm, less than 1500 mm, less than 1450 mm, less than1400 mm, less than 1350 mm, less than 1300 mm, less than 1250 mm, lessthan 1200 mm, less than 1150 mm, less than 1100 mm, less than 1050 mm,or less than 1000 mm, and/or an average stem or stalk diameter that isgreater than 18 mm, greater than 18.5 mm, greater than 19 mm, greaterthan 19.5 mm, greater than 20 mm, greater than 20.5 mm, greater than 21mm, greater than 21.5 mm, or greater than 22 mm. Any such plant heighttrait or range that is expressed in millimeters (mm) may be convertedinto a different unit of measurement based on known conversions (e.g.,one inch is equal to 2.54 cm or 25.4 millimeters, and millimeters (mm),centimeters (cm) and meters (m) only differ by one or more powers often). Thus, any measurement provided herein is further described interms of any other comparable units of measurement according to knownand established conversions. However, the exact plant height and/or stemdiameter of a modified corn plant may depend on the environment andgenetic background. Thus, the change in plant height and/or stemdiameter of a modified corn plant may instead be described in terms of aminimum difference or percent change relative to a control plant. Amodified corn plant may further comprise at least one ear that issubstantially free of male reproductive tissues or structures or otheroff-types.

According to embodiments of the present disclosure, modified corn plantsare provided that comprise a plant height during late vegetative and/orreproductive stages of development (e.g., at R3 stage) of between 1000mm and 1800 mm, between 1000 mm and 1700 mm, between 1050 mm and 1700mm, between 1100 mm and 1700 mm, between 1150 mm and 1700 mm, between1200 mm and 1700 mm, between 1250 mm and 1700 mm, between 1300 mm and1700 mm, between 1350 mm and 1700 mm, between 1400 mm and 1700 mm,between 1450 mm and 1700 mm, between 1000 mm and 1500 mm, between 1050mm and 1500 mm, between 1100 mm and 1500 mm, between 1150 mm and 1500mm, between 1200 mm and 1500 mm, between 1250 mm and 1500 mm, between1300 mm and 1500 mm, between 1350 mm and 1500 mm, between 1400 mm and1500 mm, between 1450 mm and 1500 mm, between 1000 mm and 1600 mm,between 1100 mm and 1600 mm, between 1200 mm and 1600 mm, between 1300mm and 1600 mm, between 1350 mm and 1600 mm, between 1400 mm and 1600mm, between 1450 mm and 1600 mm, of between 1000 mm and 2000 mm, between1200 mm and 2000 mm, between 1200 mm and 1800 mm, between 1300 mm and1700 mm, between 1400 mm and 1700 mm, between 1400 mm and 1600 mm,between 1400 mm and 1700 mm, between 1400 mm and 1800 mm, between 1400mm and 1900 mm, between 1400 mm and 2000 mm, or between 1200 mm and 2500mm, and/or an average stem diameter of between 17.5 mm and 22 mm,between 18 mm and 22 mm, between 18.5 and 22 mm, between 19 mm and 22mm, between 19.5 mm and 22 mm, between 20 mm and 22 mm, between 20.5 mmand 22 mm, between 21 mm and 22 mm, between 21.5 mm and 22 mm, between17.5 mm and 21 mm, between 17.5 mm and 20 mm, between 17.5 mm and 19 mm,between 17.5 mm and 18 mm, between 18 mm and 21 mm, between 18 mm and 20mm, or between 18 mm and 19 mm. A modified corn plant may besubstantially free of off-types, such as male reproductive tissues orstructures in one or more ears of the modified corn plant.

According to embodiments of the present disclosure, modified corn plantsare provided that have (i) a plant height that is at least 5%, at least10%, at least 15%, at least 20%, at least 25%, at least 30%, at least35%, at least 40%, at least 45%, at least 50%, at least 55%, at least60%, at least 65%, at least 70%, or at least 75% less than the height ofa wild-type or control plant, and/or (ii) a stem or stalk diameter thatis at least 5%, at least 10%, at least 15%, at least 20%, at least 25%,at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, atleast 55%, at least 60%, at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, or at least 100%greater than the stem diameter of the wild-type or control plant.According to embodiments of the present disclosure, a modified cornplant may have a reduced plant height that is no more than 20%, 25%,30%, 35%, 40%, 45%, 50%, 55%, or 60% shorter than the height of awild-type or control plant, and/or a stem or stalk diameter that is lessthan (or not more than) 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% greater than thestem or stalk diameter of a wild-type or control plant. For example, amodified plant may have (i) a plant height that is at least 10%, atleast 15%, or at least 20% less or shorter (i.e., greater than or equalto 10%, 15%, or 20% shorter), but not greater or more than 50% shorter,than a wild type or control plant, and/or (ii) a stem or stalk diameterthat is that is at least 5%, at least 10%, or at least 15% greater, butnot more than 30%, 35%, or 40% greater, than a wild type or controlplant. For clarity, the phrases “at least 20% shorter” and “greater thanor equal to 20% shorter” would exclude, for example, 10% shorter.Likewise for clarity, the phrases “not greater than 50% shorter”, “nomore than 50% shorter” and “not more than 50% shorter” would exclude 60%shorter; the phrase “at least 5% greater” would exclude 2% greater; andthe phrases “not more than 30% greater” and “no more than 30% greater”would exclude 40% greater.

According to embodiments of the present disclosure, modified corn plantsare provided that comprise a height between 5% and 75%, between 5% and50%, between 10% and 70%, between 10% and 65%, between 10% and 60%,between 10% and 55%, between 10% and 50%, between 10% and 45%, between10% and 40%, between 10% and 35%, between 10% and 30%, between 10% and25%, between 10% and 20%, between 10% and 15%, between 10% and 10%,between 10% and 75%, between 25% and 75%, between 10% and 50%, between20% and 50%, between 25% and 50%, between 30% and 75%, between 30% and50%, between 25% and 50%, between 15% and 50%, between 20% and 50%,between 25% and 45%, or between 30% and 45% less than the height of awild-type or control plant, and/or a stem or stalk diameter that isbetween 5% and 100%, between 5% and 95%, between 5% and 90%, between 5%and 85%, between 5% and 80%, between 5% and 75%, between 5% and 70%,between 5% and 65%, between 5% and 60%, between 5% and 55%, between 5%and 50%, between 5% and 45%, between 5% and 40%, between 5% and 35%,between 5% and 30%, between 5% and 25%, between 5% and 20%, between 5%and 15%, between 5% and 10%, between 10% and 100%, between 10% and 75%,between 10% and 50%, between 10% and 40%, between 10% and 30%, between10% and 20%, between 25% and 75%, between 25% and 50%, between 50% and75%, between 8% and 20%, or between 8% and 15% greater than the stem orstalk diameter of the wild-type or control plant.

According to embodiments of the present disclosure, modified corn plantsare provided that comprise an average internode length (or a minus-2internode length and/or minus-4 internode length relative to theposition of the ear) that is at least 5%, at least 10%, at least 15%, atleast 20%, at least 25%, at least 30%, at least 35%, at least 40%, atleast 45%, at least 50%, at least 55%, at least 60%, at least 65%, atleast 70%, or at least 75% less than the same or average internodelength of a wild-type or control plant. The “minus-2 internode” of acorn plant refers to the second internode below the ear of the plant,and the “minus-4 internode” of a corn plant refers to the fourthinternode below the ear of the plant According to many embodiments,modified corn plants are provided that have an average internode length(or a minus-2 internode length and/or minus-4 internode length relativeto the position of the ear) that is between 5% and 75%, between 5% and50%, between 10% and 70%, between 10% and 65%, between 10% and 60%,between 10% and 55%, between 10% and 50%, between 10% and 45%, between10% and 40%, between 10% and 35%, between 10% and 30%, between 10% and25%, between 10% and 20%, between 10% and 15%, between 10% and 10%,between 10% and 75%, between 25% and 75%, between 10% and 50%, between20% and 50%, between 25% and 50%, between 30% and 75%, between 30% and50%, between 25% and 50%, between 15% and 50%, between 20% and 50%,between 25% and 45%, or between 30% and 45% less than the same oraverage internode length of a wild-type or control plant.

According to embodiments of the present disclosure, modified corn plantsare provided that comprise an ear weight (individually or on average)that is at least 5%, at least 10%, at least 15%, at least 20%, at least25%, at least 30%, at least 35%, at least 40%, at least 45%, at least50%, at least 55%, at least 60%, at least 65%, at least 70%, at least75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least100% greater than the ear weight of a wild-type or control plant.

A modified corn plant provided herein may comprise an ear weight that isbetween 5% and 100%, between 5% and 95%, between 5% and 90%, between 5%and 85%, between 5% and 80%, between 5% and 75%, between 5% and 70%,between 5% and 65%, between 5% and 60%, between 5% and 55%, between 5%and 50%, between 5% and 45%, between 5% and 40%, between 5% and 35%,between 5% and 30%, between 5% and 25%, between 5% and 20%, between 5%and 15%, between 5% and 10%, between 10% and 100%, between 10% and 75%,between 10% and 50%, between 25% and 75%, between 25% and 50%, orbetween 50% and 75% greater than the ear weight of a wild-type orcontrol plant.

According to embodiments of the present disclosure, modified corn orcereal plants are provided that have a harvest index of at least 0.57,at least 0.58, at least 0.59, at least 0.60, at least 0.61, at least0.62, at least 0.63, at least 0.64, or at least 0.65 (or greater). Amodified corn plant may comprise a harvest index of between 0.57 and0.65, between 0.57 and 0.64, between 0.57 and 0.63, between 0.57 and0.62, between 0.57 and 0.61, between 0.57 and 0.60, between 0.57 and0.59, between 0.57 and 0.58, between 0.58 and 0.65, between 0.59 and0.65, or between 0.60 and 0.65. A modified corn plant may have a harvestindex that is at least 1%, at least 2%, at least 3%, at least 4%, atleast 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least10%, at least 11%, at least 12%, at least 13%, at least 14%, at least15%, at least 20%, at least 25%, at least 30%, at least 35%, at least40%, at least 45%, or at least 50% greater than the harvest index of awild-type or control plant. A modified corn plant may have a harvestindex that is between 1% and 45%, between 1% and 40%, between 1% and35%, between 1% and 30%, between 1% and 25%, between 1% and 20%, between1% and 15%, between 1% and 14%, between 1% and 13%, between 1% and 12%,between 1% and 11%, between 1% and 10%, between 1% and 9%, between 1%and 8%, between 1% and 7%, between 1% and 6%, between 1% and 5%, between1% and 4%, between 1% and 3%, between 1% and 2%, between 5% and 15%,between 5% and 20%, between 5% and 30%, or between 5% and 40% greaterthan the harvest index of a wild-type or control plant.

According to embodiments of the present disclosure, modified corn orcereal plants are provided that have an increase in harvestable yield ofat least 1 bushel per acre, at least 2 bushels per acre, at least 3bushels per acre, at least 4 bushels per acre, at least 5 bushels peracre, at least 6 bushels per acre, at least 7 bushels per acre, at least8 bushels per acre, at least 9 bushels per acre, or at least 10 bushelsper acre, relative to a wild-type or control plant. A modified cornplant may have an increase in harvestable yield between 1 and 10,between 1 and 8, between 2 and 8, between 2 and 6, between 2 and 5,between 2.5 and 4.5, or between 3 and 4 bushels per acre. A modifiedcorn plant may have an increase in harvestable yield that is at least1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, atleast 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least12%, at least 13%, at least 14%, at least 15%, at least 20%, or at least25% greater than the harvestable yield of a wild-type or control plant.A modified corn plant may have a harvestable yield that is between 1%and 25%, between 1% and 20%, between 1% and 15%, between 1% and 14%,between 1% and 13%, between 1% and 12%, between 1% and 11%, between 1%and 10%, between 1% and 9%, between 1% and 8%, between 1% and 7%,between 1% and 6%, between 1% and 5%, between 1% and 4%, between 1% and3%, between 1% and 2%, between 5% and 15%, between 5% and 20%, between5% and 25%, between 2% and 10%, between 2% and 9%, between 2% and 8%,between 2% and 7%, between 2% and 6%, between 2% and 5%, or between 2%and 4% greater than the harvestable yield of a wild-type or controlplant.

According to embodiments of the present disclosure, a modified cereal orcorn plant is provided that has a lodging frequency that is at least 5%,at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, atleast 35%, at least 40%, at least 45%, at least 50%, at least 55%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, or 100% less or lower than awild-type or control plant. A modified cereal or corn plant may have alodging frequency that is between 5% and 100%, between 5% and 95%,between 5% and 90%, between 5% and 85%, between 5% and 80%, between 5%and 75%, between 5% and 70%, between 5% and 65%, between 5% and 60%,between 5% and 55%, between 5% and 50%, between 5% and 45%, between 5%and 40%, between 5% and 35%, between 5% and 30%, between 5% and 25%,between 5% and 20%, between 5% and 15%, between 5% and 10%, between 10%and 100%, between 10% and 75%, between 10% and 50%, between 10% and 40%,between 10% and 30%, between 10% and 20%, between 25% and 75%, between25% and 50%, or between 50% and 75% less or lower than a wild-type orcontrol plant. Further provided are populations of cereal or corn plantshaving increased lodging resistance and a reduced lodging frequency.Populations of modified cereal or corn plants are provided having alodging frequency that is at least 5%, at least 10%, at least 15%, atleast 20%, at least 25%, at least 30%, at least 35%, at least 40%, atleast 45%, at least 50%, at least 55%, at least 60%, at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, or 100% less or lower than a population of wild-type orcontrol plants. A population of modified corn plants may comprise alodging frequency that is between 5% and 100%, between 5% and 95%,between 5% and 90%, between 5% and 85%, between 5% and 80%, between 5%and 75%, between 5% and 70%, between 5% and 65%, between 5% and 60%,between 5% and 55%, between 5% and 50%, between 5% and 45%, between 5%and 40%, between 5% and 35%, between 5% and 30%, between 5% and 25%,between 5% and 20%, between 5% and 15%, between 5% and 10%, between 10%and 100%, between 10% and 75%, between 10% and 50%, between 10% and 40%,between 10% and 30%, between 10% and 20%, between 25% and 75%, between25% and 50%, or between 50% and 75% less or lower than a population ofwild-type or control plants, which may be expressed as an average over aspecified number of plants or crop area of equal density.

According to embodiments of the present disclosure, modified corn plantsare provided having a significantly reduced or decreased plant height(e.g., 2000 mm or less) and a significantly increased stem diameter(e.g., 18 mm or more), relative to a wild-type or control plant.According to these embodiments, the decrease or reduction in plantheight and increase in stem diameter may be within any of the height,diameter or percentage ranges recited herein. Such modified corn plantshaving a reduced plant height and increased stem diameter relative to awild-type or control plant may be transformed with a transcribable DNAsequence encoding a non-coding RNA molecule that targets at least oneGA20 oxidase gene and/or at least one GA3 oxidase gene for suppression.Modified corn plants having a significantly reduced plant height and/ora significantly increased stem diameter relative to a wild-type orcontrol plant may further have at least one ear that is substantiallyfree of male reproductive tissues or structures and/or other off-types.Modified corn plants having a significantly reduced plant height and/oran increased stem diameter relative to a wild-type or control plant mayhave reduced activity of one or more GA20 oxidase and/or GA3 oxidasegene(s) in one or more tissue(s) of the plant, such as one or morevascular and/or leaf tissue(s) of the plant, relative to the sametissue(s) of the wild-type or control plant. According to manyembodiments, modified corn plants may comprise at least onepolynucleotide or transcribable DNA sequence encoding a non-coding RNAmolecule operably linked to a promoter, which may be a constitutive,tissue-specific or tissue-preferred promoter, wherein the non-coding RNAmolecule targets at least one GA20 oxidase and/or GA3 oxidase gene(s)for suppression as provided herein. The non-coding RNA molecule may be amiRNA, siRNA, or miRNA or siRNA precursor molecule. According to someembodiments, modified corn plants having a significantly reduced plantheight and/or an increased stem diameter relative to a wild-type orcontrol plant may further have an increased harvest index and/orincreased lodging resistance relative to the wild-type or control plant.

Modified corn or cereal plants having a significantly reduced plantheight and/or a significantly increased stem diameter relative to awild-type or control plant may comprise a mutation (e.g., an insertion,deletion, substitution, etc.) in a GA oxidase gene introduced through agene editing technology or other mutagenesis technique, whereinexpression of the GA oxidase gene is reduced or eliminated in one ormore tissues of the modified plant. Such modified corn plants having areduced plant height and/or an increased stem diameter relative to awild-type or control plant may further have an increased harvest indexand/or increased lodging resistance relative to the wild-type or controlplant. Such modified corn plants may be substantially free of off-types,such as male reproductive tissues or structures and/or other off-typesin at least one ear of the modified plants. Plant mutagenesis techniques(excluding genome editing) may include chemical mutagenesis (i.e.,treatment with a chemical mutagen, such as an azide, hydroxylamine,nitrous acid, acridine, nucleotide base analog, or alkylatingagent—e.g., EMS (ethylmethane sulfonate), MNU (N-methyl-N-nitrosourea),etc.), physical mutagenesis (e.g., gamma rays, X-rays, UV, ion beam,other forms of radiation, etc.), and insertional mutagenesis (e.g.,transposon or T-DNA insertion). Plants or various plant parts, planttissues or plant cells may be subjected to mutagenesis. Treated plantsmay be reproduced to collect seeds or produce a progeny plant, andtreated plant parts, plant tissues or plant cells may be developed orregenerated into plants or other plant tissues. Mutations generated withchemical or physical mutagenesis techniques may include a frameshift,missense or nonsense mutation leading to loss of function or expressionof a targeted gene, such as a GA3 or GA20 oxidase gene.

One method for mutagenesis of a gene is called “TILLING” (for targetinginduced local lesions in genomes), in which mutations are created in aplant cell or tissue, preferably in the seed, reproductive tissue orgermline of a plant, for example, using a mutagen, such as an EMStreatment. The resulting plants are grown and self-fertilized, and theprogeny are used to prepare DNA samples. PCR amplification andsequencing of a nucleic acid sequence of a GA oxidase gene may be usedto identify whether a mutated plant has a mutation in the GA oxidasegene. Plants having mutations in the GA oxidase gene may then be testedfor an altered trait, such as reduced plant height. Alternatively,mutagenized plants may be tested for an altered trait, such as reducedplant height, and then PCR amplification and sequencing of a nucleicacid sequence of a GA oxidase gene may be used to determine whether aplant having the altered trait also has a mutation in the GA oxidasegene. See, e.g., Colbert et al., 2001, Plant Physiol 126:480-484; andMcCallum et al., 2000, Nature Biotechnology 18:455-457. TILLING can beused to identify mutations that alter the expression a gene or theactivity of proteins encoded by a gene, which may be used to introduceand select for a targeted mutation in a GA oxidase gene of a corn orcereal plant.

Corn or cereal plants that have been subjected to a mutagenesis orgenome editing treatment may be screened and selected based on anobservable phenotype (e.g., any phenotype described herein, such asshorter plant height, increased stem/stalk diameter, etc.), or using aselection agent with a selectable marker (e.g., herbicide, etc.), ascreenable marker, or a molecular technique (e.g., lower GA levels,lower GA oxidase transcript or protein levels, presence of transgene ortranscribable sequence, etc.). Such screening and/or selectingtechniques may be used to identify and select plants having a mutationin a GA oxidase gene that leads to a desirable plant phenotype.

According to embodiments of the present disclosure, a population ofmodified corn or cereal plants are provided, wherein the population ofmodified corn or cereal plants have an average plant height that issignificantly less, and/or an average stem or stalk diameter that issignificantly more, than a population of wild-type or control plants.The population of modified corn or cereal plants may share ancestry witha single modified corn or cereal plant and/or have a single transgenicGA oxidase suppression construct insertion, event or edit in common.Modified corn plants within a population of modified corn plants maygenerally comprise at least one ear that is substantially free of malereproductive tissues or structures and/or other off-types. A populationof modified corn or cereal plants may have increased lodging resistanceon average or per number of plants or field area than a population ofwild-type or control plants. The population of modified corn or cerealplants may have a lodging frequency that is at least 5%, at least 10%,at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, atleast 70% at least 80%, at least 90%, or 100% less (or lower) than apopulation of control corn or cereal plants. A population of modifiedcorn plants may have a harvest index of at least 0.57 or greater.

According to embodiments of the present invention, modified corn orcereal plants are provided having a reduced gibberellin content (inactive form) in at least the stem and internode tissue(s), such as thestem, internode, leaf and/or vascular tissue(s), as compared to the sametissue(s) of wild-type or control plants. According to many embodiments,modified corn or cereal plants are provided having a significantlyreduced plant height and/or a significantly increased stem diameterrelative to wild-type or control plants, wherein the modified corn orcereal plants further have significantly reduced or decreased level(s)of active gibberellins or active GAs (e.g., one or more of GA1, GA3,GA4, and/or GA7) in one or more stem, internode, leaf and/or vasculartissue(s), relative to the same tissue(s) of the wild-type or controlplants. For example, the level of one or more active GAs in the stem,internode, leaf and/or vascular tissue(s) of a modified corn or cerealplant may be at least 5%, at least 10%, at least 15%, at least 20%, atleast 25%, at least 30%, at least 35%, at least 40%, at least 45%, atleast 50%, at least 55%, at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, or atleast 100% less or lower than in the same tissue(s) of a wild-type orcontrol corn plant.

According to some embodiments, a modified corn or cereal plant maycomprise an active gibberellin (GA) level(s) (e.g., one or more of GA1,GA3, GA4, and/or GA7) in one or more stem, internode, leaf and/orvascular tissue(s) that is between 5% and 50%, between 10% and 100%,between 20% and 100%, between 30% and 100%, between 40% and 100%,between 50% and 100%, between 60% and 100%, between 70% and 100%,between 80% and 100%, between 80% and 90%, between 10% and 90%, between10% and 80%, between 10% and 70%, between 10% and 60%, between 10% and50%, between 10% and 40%, between 10% and 30%, between 10% and 20%,between 50% and 100%, between 20% and 90%, between 20% and 80%, between20% and 70%, between 20% and 60%, between 20% and 50%, between 20% and40%, between 20% and 40%, between 20% and 30%, between 30% and 90%,between 30% and 80%, between 30% and 70%, between 30% and 60%, between30% and 50%, between 30% and 40%, between 40% and 90% between 40% and80%, between 40% and 70%, between 40% and 60%, between 40% and 50%,between 50% and 90%, between 50% and 80%, between 50% and 70%, between50% and 60%, between 60% and 90%, between 60% and 80%, between 60% and70%, between 70% and 90%, or between 70% and 80% less or (or lower) thanin the same tissue(s) of a wild-type or control corn plant. A modifiedcorn or cereal plant having a reduced active gibberellin (GA) level(s)in one or more stem, internode, leaf and/or vascular tissue(s) mayfurther be substantially free of off-types, such as male reproductivetissues or structures and/or other off-types in at least one ear of amodified corn plant.

According to embodiments of the present disclosure, modified corn orcereal plants are provided having a significantly reduced or eliminatedexpression level of one or more GA3 oxidase and/or GA20 oxidase genetranscript(s) and/or protein(s) in one or more tissue(s), such as one ormore stem, internode, leaf and/or vascular tissue(s), of the modifiedplants, as compared to the same tissue(s) of wild-type or controlplants. According to many embodiments, a modified corn or cereal plantis provided comprising a significantly reduced plant height and/or asignificantly increased stem diameter relative to wild-type or controlplants, wherein the modified corn or cereal plant has a significantlyreduced or eliminated expression level of one or more GA20 oxidaseand/or GA3 oxidase gene transcript(s) and/or protein(s) in one or moretissues, such as one or more stem, internode, leaf and/or vasculartissue(s), of the modified plant, as compared to the same tissue(s) of awild-type or control corn plant. For example, a modified corn or cerealplant has a significantly reduced or eliminated expression level of aGA20 oxidase_3 and/or GA20 oxidase_5 gene transcript(s) and/orprotein(s), and/or a significantly reduced or eliminated expressionlevel of a GA3 oxidase_1 and/or GA3 oxidase_2 gene transcript(s) and/orprotein(s), in one or more stem, internode, leaf and/or vasculartissue(s) of the modified plant, as compared to the same tissue(s) of awild-type or control plant. For example, the level of one or more GA3oxidase and/or GA20 oxidase gene transcript(s) and/or protein(s), or oneor more GA oxidase (or GA oxidase-like) gene transcript(s) and/orprotein(s), in one or more stem, internode, leaf and/or vasculartissue(s) of a modified corn plant may be at least 5%, at least 10%, atleast 15%, at least 20%, at least 25%, at least 30%, at least 35%, atleast 40%, at least 45%, at least 50%, at least 55%, at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, or at least 100% less or lower than in the sametissue(s) of a wild-type or control corn or cereal plant.

According to some embodiments, a modified corn or cereal plant maycomprise level(s) of one or more GA3 oxidase and/or GA20 oxidase genetranscript(s) and/or protein(s), or one or more GA oxidase (or GAoxidase-like) gene transcript(s) and/or protein(s), in one or more stem,internode, leaf and/or vascular tissue(s) that is between 5% and 50%,between 10% and 100%, between 20% and 100%, between 30% and 100%,between 40% and 100%, between 50% and 100%, between 60% and 100%,between 70% and 100%, between 80% and 100%, between 80% and 90%, between10% and 90%, between 10% and 80%, between 10% and 70%, between 10% and60%, between 10% and 50%, between 10% and 40%, between 10% and 30%,between 10% and 20%, between 50% and 100%, between 20% and 90%, between20% and 80%, between 20% and 70%, between 20% and 60%, between 20% and50%, between 20% and 40%, between 20% and 40%, between 20% and 30%,between 30% and 90%, between 30% and 80%, between 30% and 70%, between30% and 60%, between 30% and 50%, between 30% and 40%, between 40% and90% between 40% and 80%, between 40% and 70%, between 40% and 60%,between 40% and 50%, between 50% and 90%, between 50% and 80%, between50% and 70%, between 50% and 60%, between 60% and 90%, between 60% and80%, between 60% and 70%, between 70% and 90%, or between 70% and 80%less or lower than in the same tissue(s) of a wild-type or control cornor cereal plant. A modified corn or cereal plant having a reduced oreliminated expression level of at least one GA20 oxidase and/or GA3oxidase gene(s) in one or more tissue(s), may also be substantially freeof off-types, such as male reproductive tissues or structures and/orother off-types in at least one ear of the modified corn plant.

According to some embodiments, methods are provided comprising reducingor eliminating the expression of at least one GA20 oxidase gene and/orat least one GA3 oxidase gene in a crop plant, such as in one or morestem, internode, vascular and/or leaf tissue of the crop plant, whereinthe expression of the at least one GA20 oxidase gene and/or at least oneGA3 oxidase gene(s) is/are not significantly altered or changed in atleast one reproductive tissue of the plant, and/or wherein the level(s)of one or more active GAs is/are not significantly altered or changed inat least one reproductive tissue of the plant, as compared to awild-type or control plant. According to many embodiments, theexpression level(s) of at least one GA20 oxidase or GA3 oxidase gene isreduced or eliminated in at least one tissue of a modified plant with arecombinant DNA construct comprising a transcribable DNA sequenceencoding a suppression element for the GA20 oxidase or GA3 oxidase gene,such as at least one mature miRNA or miRNA precursor that is processedinto a mature miRNA, wherein the miRNA is able to reduce or suppress theexpression level of the at least one GA20 oxidase or GA3 oxidase gene,and wherein the transcribable DNA sequence is operably linked to aconstitutive, tissue-specific or tissue-preferred promoter.

Methods and techniques are provided for screening for, and/oridentifying, cells or plants, etc., for the presence of targeted editsor transgenes, and selecting cells or plants comprising targeted editsor transgenes, which may be based on one or more phenotypes or traits,or on the presence or absence of a molecular marker or polynucleotide orprotein sequence in the cells or plants. Nucleic acids can be isolatedand detected using techniques known in the art. For example, nucleicacids can be isolated and detected using, without limitation,recombinant nucleic acid technology, and/or the polymerase chainreaction (PCR). General PCR techniques are described, for example in PCRPrimer: A Laboratory Manual, Dieffenbach & Dveksler, Eds., Cold SpringHarbor Laboratory Press, 1995. Recombinant nucleic acid techniquesinclude, for example, restriction enzyme digestion and ligation, whichcan be used to isolate a nucleic acid. Isolated nucleic acids also canbe chemically synthesized, either as a single nucleic acid molecule oras a series of oligonucleotides. Polypeptides can be purified fromnatural sources (e.g., a biological sample) by known methods such asDEAE ion exchange, gel filtration, and hydroxyapatite chromatography. Apolypeptide also can be purified, for example, by expressing a nucleicacid in an expression vector. In addition, a purified polypeptide can beobtained by chemical synthesis. The extent of purity of a polypeptidecan be measured using any appropriate method, e.g., columnchromatography, polyacrylamide gel electrophoresis, or HPLC analysis.Any method known in the art may be used to screen for, and/or identify,cells, plants, etc., having a transgene or genome edit in its genome,which may be based on any suitable form of visual observation,selection, molecular technique, etc.

In some embodiments, methods are provided for detecting recombinantnucleic acids and/or polypeptides in plant cells. For example, nucleicacids may be detected using hybridization probes or through productionof amplicons using PCR with primers as known in the art. Hybridizationbetween nucleic acids is discussed in Sambrook et al. (1989, MolecularCloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y.). Polypeptides can be detected usingantibodies. Techniques for detecting polypeptides using antibodiesinclude enzyme linked immunosorbent assays (ELISAs), Western blots,immunoprecipitations, immunofluorescence, and the like. An antibodyprovided herein may be a polyclonal antibody or a monoclonal antibody.An antibody having specific binding affinity for a polypeptide providedherein can be generated using methods known in the art. An antibody orhybridization probe may be attached to a solid support, such as a tube,plate or well, using methods known in the art.

Detection (e.g., of an amplification product, of a hybridizationcomplex, of a polypeptide) can be accomplished using detectable labelsthat may be attached or associated with a hybridization probe orantibody. The term “label” is intended to encompass the use of directlabels as well as indirect labels. Detectable labels include enzymes,prosthetic groups, fluorescent materials, luminescent materials,bioluminescent materials, and radioactive materials.

The screening and selection of modified, edited or transgenic plants orplant cells can be through any methodologies known to those skilled inthe art of molecular biology. Examples of screening and selectionmethodologies include, but are not limited to, Southern analysis, PCRamplification for detection of a polynucleotide, Northern blots, RNaseprotection, primer-extension, RT-PCR amplification for detecting RNAtranscripts, Sanger sequencing, Next Generation sequencing technologies(e.g., Illumina®, PacBio®, Ion Torrent™, etc.) enzymatic assays fordetecting enzyme or ribozyme activity of polypeptides andpolynucleotides, and protein gel electrophoresis, Western blots,immunoprecipitation, and enzyme-linked immunoassays to detectpolypeptides. Other techniques such as in situ hybridization, enzymestaining, and immunostaining also can be used to detect the presence orexpression of polypeptides and/or polynucleotides. Methods forperforming all of the referenced techniques are known in the art.

Embodiments

The following paragraphs list a subset of exemplary embodiments.

Embodiment 1. A recombinant DNA construct comprising a transcribable DNAsequence encoding a non-coding RNA molecule, wherein the non-coding RNAmolecule comprises a sequence that is at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or100% complementary to at least 15, at least 16, at least 17, at least18, at least 19, at least 20, at least 21, at least 22, at least 23, atleast 24, at least 25, at least 26, or at least 27 consecutivenucleotides of a mRNA molecule encoding an endogenous GA20 oxidaseprotein in a cereal plant or plant cell, the endogenous GA20 oxidaseprotein being at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or100% identical to SEQ ID NO: 9, and wherein the transcribable DNAsequence is operably linked to a plant-expressible promoter.

Embodiment 2. The recombinant DNA construct of Embodiment 1, wherein thenon-coding RNA molecule comprises a sequence that is at least 90%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, atleast 99.5%, or 100% complementary to at least 15, at least 16, at least17, at least 18, at least 19, at least 20, at least 21, at least 22, atleast 23, at least 24, at least 25, at least 26, or at least 27consecutive nucleotides of SEQ ID NO: 7 or SEQ ID NO: 8.

Embodiment 3. The recombinant DNA construct of Embodiment 1, wherein thenon-coding RNA molecule comprises a sequence that is at least 90%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, atleast 99.5%, or 100% complementary to at least 15, at least 16, at least17, at least 18, at least 19, at least 20, at least 21, at least 22, atleast 23, at least 24, at least 25, at least 26, or at least 27consecutive nucleotides of a mRNA molecule encoding an endogenous GA20oxidase protein in a monocot or cereal plant or plant cell, theendogenous GA20 oxidase protein being at least 80%, at least 85%, atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, at least 99.5%, or 100% identical to SEQ ID NO: 15.

Embodiment 4. The recombinant DNA construct of Embodiment 3, wherein thenon-coding RNA molecule comprises a sequence that is at least 90%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, atleast 99.5%, or 100% complementary to at least 15, at least 16, at least17, at least 18, at least 19, at least 20, at least 21, at least 22, atleast 23, at least 24, at least 25, at least 26, or at least 27consecutive nucleotides of SEQ ID NO: 13 or SEQ ID NO: 14.

Embodiment 5. The recombinant DNA construct of Embodiment 1, wherein theplant-expressible promoter is a vascular promoter.

Embodiment 6. The recombinant DNA construct of Embodiment 5, wherein thevascular promoter comprises one of the following: a sucrose synthasepromoter, a sucrose transporter promoter, a Sh1 promoter, Commelinayellow mottle virus (CoYMV) promoter, a wheat dwarf geminivirus (WDV)large intergenic region (LIR) promoter, a maize streak geminivirus (MSV)coat protein (CP) promoter, a rice yellow stripe 1 (YS1)-like promoter,or a rice yellow stripe 2 (OsYSL2) promoter.

Embodiment 7. The recombinant DNA construct of Embodiment 5, wherein thevascular promoter comprises a DNA sequence that is at least 80%, atleast 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, at least 99.5% or 100% identical to one or moreof SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, or SEQ IDNO: 71, or a functional portion thereof.

Embodiment 8. The recombinant DNA construct of Embodiment 1, wherein theplant-expressible promoter is a RTBV promoter.

Embodiment 9. The recombinant DNA construct of Embodiment 8, wherein theplant-expressible promoter comprises a DNA sequence that is at least80%, at least 85%, at least 90%, at least 95%, at least 96%, at least97%, at least 98%, at least 99%, at least 99.5% or 100% identical to oneor more of SEQ ID NO: 65 or SEQ ID NO: 66, or a functional portionthereof.

Embodiment 10. The recombinant DNA construct of Embodiment 1, whereinthe plant-expressible promoter is a leaf promoter.

Embodiment 11. The recombinant DNA construct of Embodiment 10, whereinthe leaf promoter comprises one of the following: a RuBisCO promoter, aPPDK promoter, a FDA promoter, a Nadh-Gogat promoter, a chlorophyll a/bbinding protein gene promoter, a phosphoenolpyruvate carboxylase (PEPC)promoter, or a Myb gene promoter.

Embodiment 12. The recombinant DNA construct of Embodiment 10, whereinthe leaf promoter comprises a DNA sequence that is at least 80%, atleast 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, at least 99.5% or 100% identical to one or moreof SEQ ID NO: 72, SEQ ID NO: 73 or SEQ ID NO: 74, or a functionalportion thereof.

Embodiment 13. The recombinant DNA construct of Embodiment 1, whereinthe plant-expressible promoter is a constitutive promoter.

Embodiment 14. The recombinant DNA construct of Embodiment 13, whereinthe constitutive promoter is selected from the group consisting of: anactin promoter, a CaMV 35S or 19S promoter, a plant ubiquitin promoter,a plant Gos2 promoter, a FMV promoter, a CMV promoter, a MMV promoter, aPCLSV promoter, an Emu promoter, a tubulin promoter, a nopaline synthasepromoter, an octopine synthase promoter, a mannopine synthase promoter,or a maize alcohol dehydrogenase, or a functional portion thereof.

Embodiment 15. The recombinant DNA construct of Embodiment 13, whereinthe constitutive promoter comprises a DNA sequence that is at least 80%,at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, at least 99.5% or 100% identical to one or moreof SEQ ID NOs: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ IDNO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82 or SEQ ID NO: 83, ora functional portion thereof.

Embodiment 16. The recombinant DNA construct of Embodiment 1, whereinthe non-coding RNA molecule encoded by the transcribable DNA sequence isa precursor miRNA or siRNA that is processed or cleaved in a plant cellto form a mature miRNA or siRNA.

Embodiment 17. A transformation vector comprising the recombinant DNAconstruct of Embodiment 1.

Embodiment 18. A transgenic cereal plant, plant part or plant cellcomprising the recombinant DNA construct of Embodiment 1.

Embodiment 19. The transgenic cereal plant of Embodiment 18, wherein thetransgenic plant has one or more of the following traits relative to acontrol plant: shorter plant height, increased stalk/stem diameter,improved lodging resistance, deeper roots, increased leaf area, earliercanopy closure, higher stomatal conductance, lower ear height, increasedfoliar water content, improved drought tolerance, improved nitrogen useefficiency, reduced anthocyanin content and area in leaves under normalor nitrogen-limiting or water-limiting stress conditions, increased earweight, increased harvest index, increased yield, increased seed number,increased seed weight, and/or increased prolificacy.

Embodiment 20. The transgenic cereal plant of Embodiment 18, wherein thetransgenic plant has a shorter plant height and/or improved lodgingresistance.

Embodiment 21. The transgenic cereal plant of Embodiment 18, wherein theheight of the transgenic plant is at least 10%, at least 20%, at least25%, at least 30%, at least 35%, or at least 40% shorter than awild-type control plant.

Embodiment 22. The transgenic cereal plant of Embodiment 18, wherein thestalk or stem diameter of the transgenic plant at one or more steminternodes is at least 5%, at least 10%, to at least 15%, at least 20%,at least 25%, at least 30%, at least 35%, or at least 40% greater thanthe stalk or stem diameter at the same one or more internodes of awild-type control plant.

Embodiment 23. The transgenic cereal plant of any one of Embodiments 18,wherein the transgenic cereal plant is a corn plant, and wherein thestalk or stem diameter of the transgenic corn plant at one or more ofthe first, second, third, and/or fourth internode below the ear is atleast 5%, at least 10%, at least 15%, at least 20%, at least 25%, atleast 30%, at least 35%, or at least 40% greater than the same internodeof a wild-type control plant.

Embodiment 24. The transgenic cereal plant of Embodiment 18, wherein thelevel of one or more active GAs in at least one internode tissue of thestem or stalk of the transgenic plant is lower than the same internodetissue of a wild-type control plant.

Embodiment 25. The transgenic cereal plant of Embodiment 18, wherein thelevel of one or more active GAs in at least one internode tissue of thestem or stalk of the transgenic plant is at least 5%, at least 10%, atleast 15%, at least 20%, at least 25%, at least 30%, at least 35%, or atleast 40% lower than the same internode tissue of a wild-type controlplant.

Embodiment 26. A transgenic corn plant, plant part or plant cellcomprising the recombinant DNA construct of Embodiment 1.

Embodiment 27. A method for producing a transgenic cereal plant,comprising: (a) transforming at least one cell of an explant with therecombinant DNA construct of Embodiment 1, and (b) regenerating ordeveloping the transgenic cereal plant from the transformed explant.

Embodiment 28. The method of Embodiment 25, wherein the cereal plant istransformed via Agrobacterium mediated transformation or particlebombardment.

Embodiment 29. A recombinant DNA construct comprising a transcribableDNA sequence encoding a non-coding RNA molecule, wherein the non-codingRNA molecule comprises a sequence that is at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or100% complementary to at least 15, at least 16, at least 17, at least18, at least 19, at least 20, at least 21, at least 22, at least 23, atleast 24, at least 25, at least 26, or at least 27 consecutivenucleotides of a mRNA molecule encoding an endogenous GA20 oxidaseprotein in a monocot or cereal plant or plant cell, the endogenous GA20oxidase protein being at least 80%, at least 85%, at least 90%, at least95%, at least 96%, at least 97%, at least 98%, at least 99%, at least99.5%, or 100% identical to SEQ ID NO: 15, and wherein the transcribableDNA sequence is operably linked to a plant-expressible promoter.

Embodiment 30. The recombinant DNA construct of Embodiment 29, whereinthe non-coding RNA molecule comprises a sequence that is at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, atleast 99.5%, or 100% complementary to at least 15, at least 16, at least17, at least 18, at least 19, at least 20, at least 21, at least 22, atleast 23, at least 24, at least 25, at least 26, or at least 27consecutive nucleotides of SEQ ID NO: 13 or SEQ ID NO: 14.

Embodiment 31. The recombinant DNA construct of Embodiment 29, whereinthe plant-expressible promoter is a vascular promoter.

Embodiment 32. The recombinant DNA construct of Embodiment 31, whereinthe vascular promoter comprises one of the following: a sucrose synthasepromoter, a sucrose transporter promoter, a Sh1 promoter, Commelinayellow mottle virus (CoYMV) promoter, a wheat dwarf geminivirus (WDV)large intergenic region (LIR) promoter, a maize streak geminivirus (MSV)coat protein (CP) promoter, a rice yellow stripe 1 (YS1)-like promoter,or a rice yellow stripe 2 (OsYSL2) promoter.

Embodiment 33. The recombinant DNA construct of Embodiment 31, whereinthe vascular promoter comprises a DNA sequence that is at least 80%, atleast 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, at least 99.5% or 100% identical to one or moreof SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, or SEQ IDNO: 71, or a functional portion thereof.

Embodiment 34. The recombinant DNA construct of Embodiment 29, whereinthe plant-expressible promoter is a RTBV promoter.

Embodiment 35. The recombinant DNA construct of Embodiment 34, whereinthe plant-expressible promoter comprises a DNA sequence that is at least80%, at least 85%, at least 90%, at least 95%, at least 96%, at least97%, at least 98%, at least 99%, at least 99.5% or 100% identical to oneor more of SEQ ID NO: 65 or SEQ ID NO: 66, or a functional portionthereof.

Embodiment 36. The recombinant DNA construct of Embodiment 29, whereinthe plant-expressible promoter is a leaf promoter.

Embodiment 37. The recombinant DNA construct of Embodiment 36, whereinthe leaf promoter comprises one of the following: a RuBisCO promoter, aPPDK promoter, a FDA promoter, a Nadh-Gogat promoter, a chlorophyll a/bbinding protein gene promoter, a phosphoenolpyruvate carboxylase (PEPC)promoter, or a Myb gene promoter.

Embodiment 38. The recombinant DNA construct of Embodiment 36, whereinthe leaf promoter comprises a DNA sequence that is at least 80%, atleast 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, at least 99.5% or 100% identical to one or moreof SEQ ID NO: 72, SEQ ID NO: 73 or SEQ ID NO: 74, or a functionalportion thereof.

Embodiment 39. The recombinant DNA construct of Embodiment 29, whereinthe plant-expressible promoter is a constitutive promoter.

Embodiment 40. The recombinant DNA construct of Embodiment 39, whereinthe constitutive promoter is selected from the group consisting of: anactin promoter, a CaMV 35S or 19S promoter, a plant ubiquitin promoter,a plant Gos2 promoter, a FMV promoter, a CMV promoter, a MMV promoter, aPCLSV promoter, an Emu promoter, a tubulin promoter, a nopaline synthasepromoter, an octopine synthase promoter, a mannopine synthase promoter,or a maize alcohol dehydrogenase, or a functional portion thereof.

Embodiment 41. The recombinant DNA construct of Embodiment 39, whereinthe constitutive promoter comprises a DNA sequence that is at least 80%,at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, at least 99.5% or 100% identical to one or moreof SEQ ID NOs: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ IDNO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82 or SEQ ID NO: 83, ora functional portion thereof.

Embodiment 42. The recombinant DNA construct of Embodiment 29, whereinthe non-coding RNA molecule encoded by the transcribable DNA sequence isa precursor miRNA or siRNA that is processed or cleaved in a plant cellto form a mature miRNA or siRNA.

Embodiment 43. A transformation vector comprising the recombinant DNAconstruct of Embodiment 29.

Embodiment 44. A transgenic cereal plant, plant part or plant cellcomprising the recombinant DNA construct of Embodiment 29.

Embodiment 45. The transgenic cereal plant of Embodiment 44, wherein thetransgenic plant has one or more of the following traits relative to acontrol plant: shorter plant height, increased stalk/stem diameter,improved lodging resistance, deeper roots, increased leaf area, earliercanopy closure, higher stomatal conductance, lower ear height, increasedfoliar water content, improved drought tolerance, improved nitrogen useefficiency, reduced anthocyanin content and area in leaves under normalor nitrogen-limiting or water-limiting stress conditions, increased earweight, increased harvest index, increased yield, increased seed number,increased seed weight, and/or increased prolificacy.

Embodiment 46. The transgenic cereal plant of Embodiment 44, wherein thetransgenic plant has a shorter plant height and/or improved lodgingresistance.

Embodiment 47. The transgenic cereal plant of Embodiment 44, wherein theheight of the transgenic plant is at least 10%, at least 20%, at least25%, at least 30%, at least 35%, or at least 40% shorter than awild-type control plant.

Embodiment 48. The transgenic cereal plant of Embodiment 44, wherein thestalk or stem diameter of the transgenic plant at one or more steminternodes is at least 5%, at least 10%, at least 15%, at least 20%, atleast 25%, at least 30%, at least 35%, or at least 40% greater than thestalk or stem diameter at the same one or more internodes of a wild-typecontrol plant.

Embodiment 49. The transgenic cereal plant of any one of Embodiments 44,wherein the transgenic cereal plant is a corn plant, and wherein thestalk or stem diameter of the transgenic corn plant at one or more ofthe first, second, third, and/or fourth internode below the ear is atleast 5%, at least 10%, at least 15%, at least 20%, at least 25%, atleast 30%, at least 35%, or at least 40% greater than the same internodeof a wild-type control plant.

Embodiment 50. The transgenic cereal plant of Embodiment 44, wherein thelevel of one or more active GAs in at least one internode tissue of thestem or stalk of the transgenic plant is lower than the same internodetissue of a wild-type control plant.

Embodiment 51. The transgenic cereal plant of Embodiment 44, wherein thelevel of one or more active GAs in at least one internode tissue of thestem or stalk of the transgenic plant is at least 5%, at least 10%, atleast 15%, at least 20%, at least 25%, at least 30%, at least 35%, or atleast 40% lower than the same internode tissue of a wild-type controlplant.

Embodiment 52. A transgenic corn plant, plant part or plant cellcomprising the recombinant DNA construct of Embodiment 29.

Embodiment 53. A method for producing a transgenic cereal plant,comprising: (a) transforming at least one cell of an explant with therecombinant DNA construct of Embodiment 29, and (b) regenerating ordeveloping the transgenic cereal plant from the transformed explant.

Embodiment 54. The method of Embodiment 29, wherein the cereal plant istransformed via Agrobacterium mediated transformation or particlebombardment.

Embodiment 55. A recombinant DNA construct comprising a transcribableDNA sequence encoding a non-coding RNA molecule, wherein the non-codingRNA molecule comprises a sequence that is at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or100% complementary to at least 15, at least 16, at least 17, at least18, at least 19, at least 20, at least 21, at least 22, at least 23, atleast 24, at least 25, at least 26, or at least 27 consecutivenucleotides of a mRNA molecule encoding an endogenous GA3 oxidaseprotein in a monocot or cereal plant or plant cell, the endogenous GA3oxidase protein being at least 80%, at least 85%, at least 90%, at least95%, at least 96%, at least 97%, at least 98%, at least 99%, at least99.5%, or 100% identical to SEQ ID NO: 30 or 33, and wherein thetranscribable DNA sequence is operably linked to a plant-expressiblepromoter.

Embodiment 56. The recombinant DNA construct of Embodiment 55, whereinthe non-coding RNA molecule comprises a sequence that is at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, atleast 99.5%, or 100% complementary to at least 15, at least 16, at least17, at least 18, at least 19, at least 20, at least 21, at least 22, atleast 23, at least 24, at least 25, at least 26, or at least 27consecutive nucleotides of SEQ ID NO: 28, 29, 31 or 32.

Embodiment 57. The recombinant DNA construct of Embodiment 55, whereinthe plant-expressible promoter is a vascular promoter.

Embodiment 58. The recombinant DNA construct of Embodiment 57, whereinthe vascular promoter comprises one of the following: a sucrose synthasepromoter, a sucrose transporter promoter, a Sh1 promoter, Commelinayellow mottle virus (CoYMV) promoter, a wheat dwarf geminivirus (WDV)large intergenic region (LIR) promoter, a maize streak geminivirus (MSV)coat protein (CP) promoter, a rice yellow stripe 1 (YS1)-like promoter,or a rice yellow stripe 2 (OsYSL2) promoter.

Embodiment 59. The recombinant DNA construct of Embodiment 57, whereinthe vascular promoter comprises a DNA sequence that is at least 80%, atleast 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, at least 99.5% or 100% identical to one or moreof SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, or SEQ IDNO: 71, or a functional portion thereof.

Embodiment 60. The recombinant DNA construct of Embodiment 55, whereinthe plant-expressible promoter is a RTBV promoter.

Embodiment 61. The recombinant DNA construct of Embodiment 60, whereinthe plant-expressible promoter comprises a DNA sequence that is at least80%, at least 85%, at least 90%, at least 95%, at least 96%, at least97%, at least 98%, at least 99%, at least 99.5% or 100% identical to oneor more of SEQ ID NO: 65 or SEQ ID NO: 66, or a functional portionthereof Embodiment 62. The recombinant DNA construct of Embodiment 55,wherein the plant-expressible promoter is a leaf promoter.

Embodiment 63. The recombinant DNA construct of Embodiment 62, whereinthe leaf promoter comprises one of the following: a RuBisCO promoter, aPPDK promoter, a FDA promoter, a Nadh-Gogat promoter, a chlorophyll a/bbinding protein gene promoter, a phosphoenolpyruvate carboxylase (PEPC)promoter, or a Myb gene promoter.

Embodiment 64. The recombinant DNA construct of Embodiment 62, whereinthe leaf promoter comprises a DNA sequence that is at least 80%, atleast 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, at least 99.5% or 100% identical to one or moreof SEQ ID NO: 72, SEQ ID NO: 73 or SEQ ID NO: 74, or a functionalportion thereof.

Embodiment 65. The recombinant DNA construct of Embodiment 55, whereinthe plant-expressible promoter is a constitutive promoter.

Embodiment 66. The recombinant DNA construct of Embodiment 65, whereinthe constitutive promoter is selected from the group consisting of: anactin promoter, a CaMV 35S or 19S promoter, a plant ubiquitin promoter,a plant Gos2 promoter, a FMV promoter, a CMV promoter, a MMV promoter, aPCLSV promoter, an Emu promoter, a tubulin promoter, a nopaline synthasepromoter, an octopine synthase promoter, a mannopine synthase promoter,or a maize alcohol dehydrogenase, or a functional portion thereof.

Embodiment 67. The recombinant DNA construct of Embodiment 65, whereinthe constitutive promoter comprises a DNA sequence that is at least 80%,at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, at least 99.5% or 100% identical to one or moreof SEQ ID NOs: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ IDNO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82 or SEQ ID NO: 83, ora functional portion thereof.

Embodiment 68. The recombinant DNA construct of Embodiment 55, whereinthe non-coding RNA molecule encoded by the transcribable DNA sequence isa precursor miRNA or siRNA that is processed or cleaved in a plant cellto form a mature miRNA or siRNA.

Embodiment 69. A transformation vector comprising the recombinant DNAconstruct of Embodiment 55.

Embodiment 70. A transgenic cereal plant, plant part or plant cellcomprising the recombinant DNA construct of Embodiment 55.

Embodiment 71. The transgenic cereal plant of Embodiment 70, wherein thetransgenic plant has one or more of the following traits relative to acontrol plant: shorter plant height, increased stalk/stem diameter,improved lodging resistance, deeper roots, increased leaf area, earliercanopy closure, higher stomatal conductance, lower ear height, increasedfoliar water content, improved drought tolerance, improved nitrogen useefficiency, reduced anthocyanin content and area in leaves under normalor nitrogen-limiting or water-limiting stress conditions, increased earweight, increased harvest index, increased yield, increased seed number,increased seed weight, and/or increased prolificacy.

Embodiment 72. The transgenic cereal plant of Embodiment 70, wherein thetransgenic plant has a shorter plant height and/or improved lodgingresistance.

Embodiment 73. The transgenic cereal plant of Embodiment 70, wherein theheight of the transgenic plant is at least 10%, at least 20%, at least25%, at least 30%, at least 35%, or at least 40% shorter than awild-type control plant.

Embodiment 74. The transgenic cereal plant of Embodiment 70, wherein thestalk or stem diameter of the transgenic plant at one or more steminternodes is at least 5%, at least 10%, at least 15%, at least 20%, atleast 25%, at least 30%, at least 35%, or at least 40% greater than thestalk or stem diameter at the same one or more internodes of a wild-typecontrol plant.

Embodiment 75. The transgenic cereal plant of any one of Embodiments 70,wherein the transgenic cereal plant is a corn plant, and wherein thestalk or stem diameter of the transgenic corn plant at one or more ofthe first, second, third, and/or fourth internode below the ear is atleast 5%, at least 10%, at least 15%, at least 20%, at least 25%, atleast 30%, at least 35%, or at least 40% greater than the same internodeof a wild-type control plant.

Embodiment 76. The transgenic cereal plant of Embodiment 70, wherein thelevel of one or more active GAs in at least one internode tissue of thestem or stalk of the transgenic plant is lower than the same internodetissue of a wild-type control plant.

Embodiment 77. The transgenic cereal plant of Embodiment 70, wherein thelevel of one or more active GAs in at least one internode tissue of thestem or stalk of the transgenic plant is at least 5%, at least 10%, atleast 15%, at least 20%, at least 25%, at least 30%, at least 35%, or atleast 40% lower than the same internode tissue of a wild-type controlplant.

Embodiment 78. A transgenic corn plant, plant part or plant cellcomprising the recombinant DNA construct of Embodiment 55.

Embodiment 79. A method for producing a transgenic cereal plant,comprising: (a) transforming at least one cell of an explant with therecombinant DNA construct of Embodiment 55, and (b) regenerating ordeveloping the transgenic cereal plant from the transformed explant.

Embodiment 80. The method of Embodiment 79, wherein the cereal plant istransformed via Agrobacterium mediated transformation or particlebombardment.

Embodiment 81. A recombinant DNA construct comprising a transcribableDNA sequence encoding a non-coding RNA molecule, wherein the non-codingRNA molecule comprises a sequence that is at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or100% complementary to at least 15, at least 16, at least 17, at least18, at least 19, at least 20, at least 21, at least 22, at least 23, atleast 24, at least 25, at least 26, or at least 27 consecutivenucleotides of a mRNA molecule encoding an endogenous GA20 oxidaseprotein in a monocot or cereal plant or plant cell, the endogenous GA20oxidase protein being at least 80%, at least 85%, at least 90%, at least95%, at least 96%, at least 97%, at least 98%, at least 99%, at least99.5%, or 100% identical to SEQ ID NO: 12, and wherein the transcribableDNA sequence is operably linked to a plant-expressible promoter.

Embodiment 82. The recombinant DNA construct of Embodiment 81, whereinthe non-coding RNA molecule comprises a sequence that is at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, atleast 99.5%, or 100% complementary to at least 15, at least 16, at least17, at least 18, at least 19, at least 20, at least 21, at least 22, atleast 23, at least 24, at least 25, at least 26, or at least 27consecutive nucleotides of SEQ ID NO: 10 or 11.

Embodiment 83. The recombinant DNA construct of Embodiment 81, whereinthe plant-expressible promoter is a vascular promoter.

Embodiment 84. The recombinant DNA construct of Embodiment 83, whereinthe vascular promoter comprises one of the following: a sucrose synthasepromoter, a sucrose transporter promoter, a Sh1 promoter, Commelinayellow mottle virus (CoYMV) promoter, a wheat dwarf geminivirus (WDV)large intergenic region (LIR) promoter, a maize streak geminivirus (MSV)coat protein (CP) promoter, a rice yellow stripe 1 (YS1)-like promoter,or a rice yellow stripe 2 (OsYSL2) promoter.

Embodiment 85. The recombinant DNA construct of Embodiment 83, whereinthe vascular promoter comprises a DNA sequence that is at least 80%, atleast 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, at least 99.5% or 100% identical to one or moreof SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, or SEQ IDNO: 71, or a functional portion thereof Embodiment 86. The recombinantDNA construct of Embodiment 81, wherein the plant-expressible promoteris a RTBV promoter.

Embodiment 87. The recombinant DNA construct of Embodiment 86, whereinthe plant-expressible promoter comprises a DNA sequence that is at least80%, at least 85%, at least 90%, at least 95%, at least 96%, at least97%, at least 98%, at least 99%, at least 99.5% or 100% identical to oneor more of SEQ ID NO: 65 or SEQ ID NO: 66, or a functional portionthereof.

Embodiment 88. The recombinant DNA construct of Embodiment 81, whereinthe plant-expressible promoter is a leaf promoter.

Embodiment 89. The recombinant DNA construct of Embodiment 88, whereinthe leaf promoter comprises one of the following: a RuBisCO promoter, aPPDK promoter, a FDA promoter, a Nadh-Gogat promoter, a chlorophyll a/bbinding protein gene promoter, a phosphoenolpyruvate carboxylase (PEPC)promoter, or a Myb gene promoter.

Embodiment 90. The recombinant DNA construct of Embodiment 88, whereinthe leaf promoter comprises a DNA sequence that is at least 80%, atleast 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, at least 99.5% or 100% identical to one or moreof SEQ ID NO: 72, SEQ ID NO: 73 or SEQ ID NO: 74, or a functionalportion thereof.

Embodiment 91. The recombinant DNA construct of Embodiment 81, whereinthe plant-expressible promoter is a constitutive promoter.

Embodiment 92. The recombinant DNA construct of Embodiment 91, whereinthe constitutive promoter is selected from the group consisting of: anactin promoter, a CaMV 35S or 19S promoter, a plant ubiquitin promoter,a plant Gos2 promoter, a FMV promoter, a CMV promoter, a MMV promoter, aPCLSV promoter, an Emu promoter, a tubulin promoter, a nopaline synthasepromoter, an octopine synthase promoter, a mannopine synthase promoter,or a maize alcohol dehydrogenase, or a functional portion thereof.

Embodiment 93. The recombinant DNA construct of Embodiment 91, whereinthe constitutive promoter comprises a DNA sequence that is at least 80%,at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, at least 99.5% or 100% identical to one or moreof SEQ ID NOs: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ IDNO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82 or SEQ ID NO: 83, ora functional portion thereof.

Embodiment 94. The recombinant DNA construct of Embodiment 81, whereinthe non-coding RNA molecule encoded by the transcribable DNA sequence isa precursor miRNA or siRNA that is processed or cleaved in a plant cellto form a mature miRNA or siRNA.

Embodiment 95. A transformation vector comprising the recombinant DNAconstruct of Embodiment 81.

Embodiment 96. A transgenic cereal plant, plant part or plant cellcomprising the recombinant DNA construct of Embodiment 81.

Embodiment 97. The transgenic cereal plant of Embodiment 96, wherein thetransgenic plant has one or more of the following traits relative to acontrol plant: shorter plant height, increased stalk/stem diameter,improved lodging resistance, deeper roots, increased leaf area, earliercanopy closure, higher stomatal conductance, lower ear height, increasedfoliar water content, improved drought tolerance, improved nitrogen useefficiency, reduced anthocyanin content and area in leaves under normalor nitrogen-limiting or water-limiting stress conditions, increased earweight, increased harvest index, increased yield, increased seed number,increased seed weight, and/or increased prolificacy.

Embodiment 98. The transgenic cereal plant of Embodiment 96, wherein thetransgenic plant has a shorter plant height and/or improved lodgingresistance.

Embodiment 99. The transgenic cereal plant of Embodiment 96, wherein theheight of the transgenic plant is at least 10%, at least 20%, at least25%, at least 30%, at least 35%, or at least 40% shorter than awild-type control plant.

Embodiment 100. The transgenic cereal plant of Embodiment 96, whereinthe stalk or stem diameter of the transgenic plant at one or more steminternodes is at least 5%, at least 10%, at least 15%, at least 20%, atleast 25%, at least 30%, at least 35%, or at least 40% greater than thestalk or stem diameter at the same one or more internodes of a wild-typecontrol plant.

Embodiment 101. The transgenic cereal plant of any one of Embodiments96, wherein the transgenic cereal plant is a corn plant, and wherein thestalk or stem diameter of the transgenic corn plant at one or more ofthe first, second, third, and/or fourth internode below the ear is atleast 5%, at least 10%, at least 15%, at least 20%, at least 25%, atleast 30%, at least 35%, or at least 40% greater than the same internodeof a wild-type control plant.

Embodiment 102. The transgenic cereal plant of Embodiment 96, whereinthe level of one or more active GAs in at least one internode tissue ofthe stem or stalk of the transgenic plant is lower than the sameinternode tissue of a wild-type control plant.

Embodiment 103. The transgenic cereal plant of Embodiment 96, whereinthe level of one or more active GAs in at least one internode tissue ofthe stem or stalk of the transgenic plant is at least 5%, at least 10%,at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, orat least 40% lower than the same internode tissue of a wild-type controlplant.

Embodiment 104. A transgenic corn plant, plant part or plant cellcomprising the recombinant DNA construct of Embodiment 81.

Embodiment 105. A method for producing a transgenic cereal plant,comprising: (a) transforming at least one cell of an explant with therecombinant DNA construct of Embodiment 81, and (b) regenerating ordeveloping the transgenic cereal plant from the transformed explant.

Embodiment 106. The method of Embodiment 105, wherein the cereal plantis transformed via Agrobacterium mediated transformation or particlebombardment.

Embodiment 107. The recombinant DNA construct of Embodiment 1, 29, 55 or81, wherein the non-coding RNA molecule comprises a sequence that is atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, at least 99.5%, or 100% complementary to at least 15, atleast 16, at least 17, at least 18, at least 19, at least 20, at least21, at least 22, at least 23, at least 24, at least 25, at least 26, orat least 27 consecutive nucleotides of a mRNA molecule encoding anendogenous GA oxidase protein in a monocot or cereal plant or plantcell, the endogenous GA oxidase protein being at least 80%, at least85%, at least 90%, at least 95%, at least 96%, at least 97%, at least98%, at least 99%, at least 99.5%, or 100% identical to one or more ofSEQ ID NOs: 3, 6, 9, 12, 15, 18, 21, 24, 27, 30 and 33.

Embodiment 108. The recombinant DNA construct of Embodiment 107, whereinthe non-coding RNA molecule comprises a sequence that is at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, atleast 99.5%, or 100% complementary to at least 15, at least 16, at least17, at least 18, at least 19, at least 20, at least 21, at least 22, atleast 23, at least 24, at least 25, at least 26, or at least 27consecutive nucleotides of one or more of SEQ ID NOs: 1, 2, 4, 5, 7, 8,10, 11, 13, 14, 16, 17, 19, 20, 22, 23, 25, 26, 28, 29, 31, and 32.

Embodiment 109. A recombinant DNA construct comprising a transcribableDNA sequence encoding a non-coding RNA molecule, wherein the non-codingRNA molecule comprises a sequence that is at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or100% complementary to at least 15, at least 16, at least 17, at least18, at least 19, at least 20, at least 21, at least 22, at least 23, atleast 24, at least 25, at least 26, or at least 27 consecutivenucleotides of a mRNA molecule encoding an endogenous protein in amonocot or cereal plant or plant cell, the endogenous protein being atleast 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, at least 99.5%, or 100% identicalto SEQ ID NO: 86, 90, 94, 97, 101, 104, 108, 112, 116, 118, 121, 125,129, 133, or 136, and wherein the transcribable DNA sequence is operablylinked to a plant-expressible promoter.

Embodiment 110. The recombinant DNA construct of Embodiment 109, whereinthe non-coding RNA molecule comprises a sequence that is at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, atleast 99.5%, or 100% complementary to at least 15, at least 16, at least17, at least 18, at least 19, at least 20, at least 21, at least 22, atleast 23, at least 24, at least 25, at least 26, or at least 27consecutive nucleotides of SEQ ID NO: 84, 85, 87, 88, 89, 91, 92, 93,95, 96, 98, 99, 100, 102, 103, 105, 106, 107, 109, 110, 111, 113, 114,115, 119, 120, 122, 123, 124, 126, 127, 128, 130, 131, 132, 134, 135, or137.

Embodiment 111. The recombinant DNA construct of Embodiment 109, whereinthe plant-expressible promoter is a vascular promoter.

Embodiment 112. The recombinant DNA construct of Embodiment 109, whereinthe plant-expressible promoter is a RTBV promoter.

Embodiment 113. The recombinant DNA construct of Embodiment 109, whereinthe plant-expressible promoter is a leaf promoter.

Embodiment 114. The recombinant DNA construct of Embodiment 109, whereinthe plant-expressible promoter is a constitutive promoter.

Embodiment 115. A transformation vector comprising the recombinant DNAconstruct of Embodiment 81.

Embodiment 116. A transgenic cereal plant, plant part or plant cellcomprising the recombinant DNA construct of Embodiment 109.

Embodiment 117. The transgenic cereal plant of Embodiment 116, whereinthe transgenic plant has a shorter plant height and/or improved lodgingresistance.

Embodiment 118. The transgenic cereal plant of Embodiment 116, whereinthe level of one or more active GAs in at least one internode tissue ofthe stem or stalk of the transgenic plant is lower than the sameinternode tissue of a wild-type control plant.

Embodiment 119. A method for producing a transgenic cereal plant,comprising: (a) transforming at least one cell of an explant with therecombinant DNA construct of Embodiment 116, and (b) regenerating ordeveloping the transgenic cereal plant from the transformed explant.

Embodiment 120. The method of Embodiment 119, wherein the cereal plantis transformed via Agrobacterium mediated transformation or particlebombardment.

Embodiment 121. A method for lowering the level of at least one activeGA molecule in the stem or stalk of a corn or cereal plant comprising:suppressing one or more GA3 oxidase or GA20 oxidase genes with arecombinant DNA construct in one or more tissues of the transgeniccereal or corn plant.

Embodiment 122. The method of Embodiment 121, wherein the recombinantDNA construct encodes a non-coding RNA molecule that targets one or moreGA3 or GA20 oxidase genes for suppression, wherein the transcribable DNAsequence is operably linked to a plant-expressible promoter.

Embodiment 123. The method of Embodiment 122, wherein theplant-expressible promoter is a vascular promoter.

Embodiment 124. The method of Embodiment 122, wherein theplant-expressible promoter is a RTBV promoter.

Embodiment 125. The method of Embodiment 122, wherein theplant-expressible promoter is a constitutive promoter.

Embodiment 126. The method of Embodiment 122, wherein theplant-expressible promoter is a leaf promoter.

Embodiment 127. The method of Embodiment 121, wherein the transgeniccorn or cereal plant is a corn plant.

Embodiment 128. A transgenic corn or cereal plant comprising arecombinant DNA construct, wherein the recombinant DNA constructcomprises a transcribable DNA sequence encoding a non-coding RNAmolecule that targets at least one endogenous GA20 or GA3 oxidase genefor suppression, the transcribable DNA sequence being operably linked toa plant-expressible promoter, and wherein the transgenic monocot orcereal plant has a shorter plant height relative to a wild-type controlplant.

Embodiment 129. The transgenic corn or cereal plant of Embodiment 128,wherein the transgenic plant has one or more of the following additionaltraits relative to the control plant: increased stalk/stem diameter,improved lodging resistance, deeper roots, increased leaf area, earliercanopy closure, higher stomatal conductance, lower ear height, increasedfoliar water content, improved drought tolerance, improved nitrogen useefficiency, reduced anthocyanin content and area in leaves under normalor nitrogen or water limiting stress conditions, increased ear weight,increased harvest index, increased yield, increased seed number,increased seed weight, and increased prolificacy.

Embodiment 130. The transgenic corn or cereal plant of Embodiment 128,wherein the height of the transgenic plant is at least 10%, at least20%, at least 25%, at least 30%, at least 35%, or at least 40% shorterthan the control plant.

Embodiment 131. The transgenic corn or cereal plant of Embodiment 128,wherein the stalk or stem diameter of the transgenic plant at one ormore stem internodes is at least 5%, at least 10%, at least 15%, atleast 20%, at least 25%, at least 30%, at least 35%, or at least 40%greater than the control plant.

Embodiment 132. The transgenic corn or cereal plant of any one ofEmbodiments 128, wherein the level of one or more active GAs in at leastone internode tissue of the stem or stalk of the transgenic plant islower than the same internode tissue of the control plant.

Embodiment 133. The transgenic corn or cereal plant of any one ofEmbodiments 128, wherein the level of one or more active GAs in at leastone internode tissue of the stem or stalk of the transgenic plant is atleast 5%, at least 10%, at least 15%, at least 20%, at least 25%, atleast 30%, at least 35%, or at least 40% lower than the same internodetissue of the control plant.

Embodiment 134. The transgenic corn or cereal plant of any one ofEmbodiments 128, wherein the transgenic plant does not have anysignificant off-types in at least one female organ or ear.

Embodiment 135. The transgenic corn or cereal plant of any one ofEmbodiments 128, wherein the transgenic cereal plant is a corn plant,and wherein the non-coding RNA molecule targets the endogenous GA20oxidase_3 and/or GA20 oxidase_5 gene(s) for suppression.

Embodiment 136. The transgenic corn or cereal plant of Embodiment 128,wherein the plant-expressible promoter is a vascular promoter.

Embodiment 137. The transgenic corn or cereal plant of Embodiment 128,wherein the plant-expressible promoter is a RTBV promoter.

Embodiment 138. The transgenic corn or cereal plant of Embodiment 128,wherein the plant-expressible promoter is a constitutive promoter.

Embodiment 139. The transgenic corn or cereal plant of Embodiment 128,wherein the plant-expressible promoter is a leaf promoter.

Embodiment 140. The transgenic corn or cereal plant of Embodiment 128,wherein the transgenic plant has one or more of the following additionaltraits relative to the control plant: increased stalk/stem diameter,improved lodging resistance, deeper roots, increased leaf area, earliercanopy closure, higher stomatal conductance, lower ear height, increasedfoliar water content, improved drought tolerance, improved nitrogen useefficiency, reduced anthocyanin content and area in leaves under normalor nitrogen or water limiting stress conditions, increased ear weight,increased harvest index, increased yield, increased seed number,increased seed weight, and increased prolificacy.

Embodiment 141. A cereal plant comprising a mutation at or near anendogenous GA oxidase gene introduced by a mutagenesis technique,wherein the expression level of the endogenous GA oxidase gene isreduced or eliminated in the cereal plant, and wherein the cereal planthas a shorter plant height relative to a wild-type control plant.

Embodiment 142. The cereal plant of Embodiment 141, wherein the cerealplant comprising the mutation has one or more of the followingadditional traits relative to the control plant: increased stalk/stemdiameter, improved lodging resistance, deeper roots, increased leafarea, earlier canopy closure, higher stomatal conductance, lower earheight, increased foliar water content, improved drought tolerance,improved nitrogen use efficiency, reduced anthocyanin content and areain leaves under normal or nitrogen or water limiting stress conditions,increased ear weight, increased harvest index, increased yield,increased seed number, increased seed weight, and increased prolificacy.

Embodiment 143. The cereal plant of Embodiment 141, wherein the heightof the cereal plant is at least 10%, at least 20%, at least 25%, atleast 30%, at least 35%, or at least 40% shorter than the control plant.

Embodiment 144. The cereal plant of Embodiment 141, wherein the stalk orstem diameter of the cereal plant at one or more stem internodes is atleast 5%, at least 10%, at least 15%, at least 20%, at least 25%, atleast 30%, at least 35%, or at least 40% greater than the control plant.

Embodiment 145. The cereal plant of Embodiment 141, wherein the level ofone or more active GAs in at least one internode tissue of the stem orstalk of the cereal plant is lower than the same internode tissue of thecontrol plant.

Embodiment 146. The cereal plant of Embodiment 141, wherein the level ofone or more active GAs in at least one internode tissue of the stem orstalk of the cereal plant is at least 5%, at least 10%, at least 15%, atleast 20%, at least 25%, at least 30%, at least 35%, or at least 40%lower than the same internode tissue of the control plant.

Embodiment 147. The cereal plant of Embodiment 141, wherein the cerealplant does not have any significant off-types in at least one femaleorgan or ear.

Embodiment 148. The cereal plant of Embodiment 141, wherein the cerealplant is a corn plant.

Embodiment 149. A corn or cereal plant comprising a genomic editintroduced via a targeted genome editing technique at or near the locusof an endogenous GA oxidase gene, wherein the expression level of theendogenous GA oxidase gene is reduced or eliminated relative to acontrol plant, and wherein the edited cereal plant has a shorter plantheight relative to the control plant.

Embodiment 150. The edited corn or cereal plant of Embodiment 149,wherein the edited plant has one or more of the following additionaltraits relative to the control plant: increased stalk/stem diameter,improved lodging resistance, deeper roots, increased leaf area, earliercanopy closure, higher stomatal conductance, lower ear height, increasedfoliar water content, improved drought tolerance, improved nitrogen useefficiency, reduced anthocyanin content and area in leaves under normalor nitrogen or water limiting stress conditions, increased ear weight,increased harvest index, increased yield, increased seed number,increased seed weight, and increased prolificacy.

Embodiment 151. The edited corn or cereal plant of Embodiment 149,wherein the height of the edited plant is at least 10%, at least 20%, atleast 25%, at least 30%, at least 35%, or at least 40% shorter than thecontrol plant.

Embodiment 152. The edited corn or cereal plant of Embodiment 149,wherein the stalk or stem diameter of the edited plant at one or morestem internodes is at least 5%, at least 10%, at least 15%, at least20%, at least 25%, at least 30%, at least 35%, or at least 40% greaterthan the control plant.

Embodiment 153. The edited corn or cereal plant of Embodiment 149,wherein the level of one or more active GAs in at least one internodetissue of the stem or stalk of the edited plant is lower than the sameinternode tissue of the control plant.

Embodiment 154. The edited corn or cereal plant of Embodiment 149,wherein the level of one or more active GAs in at least one internodetissue of the stem or stalk of the edited plant is at least 5%, at least10%, at least 15%, at least 20%, at least 25%, at least 30%, at least35%, or at least 40% lower than the same internode tissue of the controlplant.

Embodiment 155. The edited corn or cereal plant of Embodiment 149,wherein the edited plant does not have any significant off-types in atleast one female organ or ear.

Embodiment 156. The edited corn or cereal plant of Embodiment 149,wherein the genomic edit is introduced using a meganuclease, azinc-finger nuclease (ZFN), a RNA-guided endonuclease, aTALE-endonuclease (TALEN), a recombinase, or a transposase.

Embodiment 157. The edited corn or cereal plant of Embodiment 149,wherein the genomic edit comprises a substitution, deletion, insertion,or inversion of one or more nucleotides relative to the sequence of theendogenous GA oxidase gene in the control plant.

Embodiment 158. A composition comprising a guide RNA, wherein the guideRNA comprises a guide sequence that is at least 95%, at least 96%, atleast 97%, at least 99%, or 100% identical or complementary to at least10, at least 11, at least 12, at least 13, at least 14, at least 15, atleast 16, at least 17, at least 18, at least 19, at least 20, at least21, at least 22, at least 23, at least 24, or at least 25 consecutivenucleotides of a target DNA sequence at or near the genomic locus of anendogenous GA oxidase gene of a cereal plant.

Embodiment 159. The composition of Embodiment 158, wherein the guide RNAmolecule comprises a guide sequence that is at least 95%, at least 96%,at least 97%, at least 99% or 100% complementary to at least 10, atleast 11, at least 12, at least 13, at least 14, at least 15, at least16, at least 17, at least 18, at least 19, at least 20, at least 21, atleast 22, at least 23, at least 24, or at least 25 consecutivenucleotides of SEQ ID NO: 34, 35 or 38, or a sequence complementarythereto.

Embodiment 160. The composition of Embodiment 158, wherein the guide RNAmolecule comprises a guide sequence that is at least 95%, at least 96%,at least 97%, at least 99% or 100% complementary to at least 10, atleast 11, at least 12, at least 13, at least 14, at least 15, at least16, at least 17, at least 18, at least 19, at least 20, at least 21, atleast 22, at least 23, at least 24, or at least 25 consecutivenucleotides of SEQ ID NO: 87, 91, 95, 98, 105, 109, 113, 117, 122, 126,130 or 137, or a sequence complementary thereto.

Embodiment 161. The composition of Embodiment 158, further comprising anRNA-guided endonuclease.

Embodiment 162. The composition of Embodiment 161, wherein theRNA-guided endonuclease in the presence of the guide RNA molecule causesa double strand break or nick at or near the target DNA sequence in thegenome of the cereal plant.

Embodiment 163. The composition of Embodiment 161, wherein theRNA-guided endonuclease is selected from the group consisting of Cas1,Cas1B, Cas2, Cas3, Cas4, Cas5, Cash, Cas7, Cas8, Cas9, Cas10, Csy1,Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn1, Csn2, Csm2, Csm3, Csm4,Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17,Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx12, Csx15, Csf1, Csf2, Csf3,Csf4, Cpf1, CasX, CasY, Argonaute, and any homologs or modified versionsthereof having RNA-guided endonuclease activity.

Embodiment 164. The composition of Embodiment 158, further comprising arecombinant DNA donor template comprising at least one homology sequenceor homology arm, wherein the at least one homology sequence or homologyarm is at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 95%, at least 96%, at least 97%, at least 99% or 100%complementary to at least 20, at least 25, at least 30, at least 35, atleast 40, at least 45, at least 50, at least 60, at least 70, at least80, at least 90, at least 100, at least 150, at least 200, at least 250,at least 500, at least 1000, at least 2500, or at least 5000 consecutivenucleotides of a target DNA sequence, wherein the target DNA sequence isa genomic sequence at or near the genomic locus of the endogenous GAoxidase gene of a corn or cereal plant.

Embodiment 165. A recombinant DNA construct comprising a transcribableDNA sequence encoding a non-coding guide RNA molecule, wherein the guideRNA molecule comprises a guide sequence that is at least 95%, at least96%, at least 97%, at least 99% or 100% complementary to at least 10, atleast 11, at least 12, at least 13, at least 14, at least 15, at least16, at least 17, at least 18, at least 19, at least 20, at least 21, atleast 22, at least 23, at least 24, or at least 25 consecutivenucleotides of a target DNA sequence at or near the genomic locus of anendogenous GA oxidase gene of a corn or cereal plant.

Embodiment 166. The recombinant DNA construct of Embodiment 165, whereinthe guide RNA comprises a guide sequence that is at least 95%, at least96%, at least 97%, at least 99% or 100% complementary to at least 10, atleast 11, at least 12, at least 13, at least 14, at least 15, at least16, at least 17, at least 18, at least 19, at least 20, at least 21, atleast 22, at least 23, at least 24, or at least 25 consecutivenucleotides of SEQ ID NO: 34, 35 or 38, or a sequence complementarythereto.

Embodiment 167. The recombinant DNA construct of Embodiment 165, whereinthe guide RNA molecule comprises a guide sequence that is at least 95%,at least 96%, at least 97%, at least 99% or 100% complementary to atleast 10, at least 11, at least 12, at least 13, at least 14, at least15, at least 16, at least 17, at least 18, at least 19, at least 20, atleast 21, at least 22, at least 23, at least 24, or at least 25consecutive nucleotides of SEQ ID NO: 87, 91, 95, 98, 105, 109, 113,117, 122, 126, 130 or 137, or a sequence complementary thereto.

Embodiment 168. The recombinant DNA construct of Embodiment 165, whereinthe transcribable DNA sequence is operably linked to a plant-expressiblepromoter.

Embodiment 169. The recombinant DNA construct of Embodiment 165, whereinthe guide RNA molecule is a CRISPR RNA (crRNA) or a single-chain guideRNA (sgRNA).

Embodiment 170. The recombinant DNA construct of Embodiment 165, whereinthe guide RNA comprises a sequence complementary to a protospaceradjacent motif (PAM) sequence present in the genome of the cereal plantimmediately adjacent to the target DNA sequence at or near the genomiclocus of the endogenous GA oxidase gene.

Embodiment 171. The recombinant DNA construct of any one of Embodiment165, wherein the PAM sequence comprises a canonical 5′-NGG-3′ sequence.

Embodiment 172. The recombinant DNA construct of Embodiment 165, whereinthe endogenous GA oxidase gene encodes a protein that is at least 80%,at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO:9, 12 or 15.

Embodiment 173. A DNA molecule comprising the recombinant DNA constructof Embodiment 165.

Embodiment 174. A transformation vector comprising the recombinant DNAconstruct of Embodiment 165.

Embodiment 175. A bacterial cell comprising the recombinant DNAconstruct of Embodiment 165.

Embodiment 176. A corn or cereal plant, plant part or plant cellcomprising the recombinant DNA construct of Embodiment 165.

Embodiment 177. A composition comprising the recombinant DNA constructof Embodiment 165.

Embodiment 178. The composition of Embodiment 177, further comprising aRNA-guided endonuclease.

Embodiment 179. The composition of Embodiment 177, wherein theRNA-guided endonuclease is selected from the group consisting of Cas1,Cas1B, Cas2, Cas3, Cas4, Cas5, Cash, Cas7, Cas8, Cas9, Cas10, Csy1,Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5,Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14,Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, Cpf1,Argonaute, and homologs or modified versions thereof having RNA-guidedendonuclease activity.

Embodiment 180. The composition of Embodiment 177, further comprising asecond recombinant DNA construct comprising a second transcribable DNAsequence encoding the RNA-guided endonuclease.

Embodiment 181. The composition of Embodiment 177, comprising a DNAmolecule or vector comprising the recombinant DNA construct and thesecond recombinant DNA construct.

Embodiment 182. The composition of Embodiment 177, comprising a firstDNA molecule or vector and a second DNA molecule or vector, wherein thefirst DNA molecule or vector comprises the recombinant DNA constructencoding the guide RNA molecule, and the second DNA molecule or vectorcomprises the second recombinant DNA construct encoding the RNA-guidedendonuclease.

Embodiment 183. The composition of Embodiment 177, further comprising arecombinant DNA donor template comprising at least one homology sequenceor homology arm, wherein the at least one homology sequence or homologyarm is at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 95%, at least 96%, at least 97%, at least 99% or 100%complementary to at least 20, at least 25, at least 30, at least 35, atleast 40, at least 45, at least 50, at least 60, at least 70, at least80, at least 90, at least 100, at least 150, at least 200, at least 250,at least 500, at least 1000, at least 2500, or at least 5000 consecutivenucleotides of a target DNA sequence, wherein the target DNA sequence isa genomic sequence at or near the genomic locus of an endogenous GAoxidase gene of a corn or cereal plant.

Embodiment 184. A recombinant DNA donor template comprising at least onehomology sequence, wherein the at least one homology sequence is atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, at least 96%, at least 97%, at least 99% or 100%complementary to at least 20, at least 25, at least 30, at least 35, atleast 40, at least 45, at least 50, at least 60, at least 70, at least80, at least 90, at least 100, at least 150, at least 200, at least 250,at least 500, at least 1000, at least 2500, or at least 5000 consecutivenucleotides of a target DNA sequence, wherein the target DNA sequence isa genomic sequence at or near the genomic locus of an endogenous GAoxidase gene of a corn or cereal plant.

Embodiment 185. The recombinant DNA donor template of Embodiment 184,wherein the at least one homology sequence comprises at least onemutation relative to the complementary strand of the target DNA sequenceat or near the genomic locus of the endogenous GA oxidase gene.

Embodiment 186. The recombinant DNA donor template of Embodiment 185,wherein the at least one mutation comprises a substitution, deletion,insertion, or inversion of one or more nucleotides relative to thecomplementary strand of the target DNA sequence.

Embodiment 187. The recombinant DNA donor template of Embodiment 184,wherein the at least one homology sequence is at least 70%, at least75%, at least 80%, at least 85%, at least 90%, at least 95%, at least96%, at least 97%, at least 99% or 100% identical or complementary to atleast 20, at least 25, at least 30, at least 35, at least 40, at least45, at least 50, at least 60, at least 70, at least 80, at least 90, atleast 100, at least 150, at least 200, at least 250, at least 500, atleast 1000, at least 2500, or at least 5000 consecutive nucleotides ofSEQ ID NO: 34, 35 or 38, or a sequence complementary thereto.

Embodiment 188. The recombinant DNA donor template of Embodiment 184,wherein the at least one homology sequence is at least 70%, at least75%, at least 80%, at least 85%, at least 90%, at least 95%, at least96%, at least 97%, at least 99% or 100% identical or complementary to atleast 20, at least 25, at least 30, at least 35, at least 40, at least45, at least 50, at least 60, at least 70, at least 80, at least 90, atleast 100, at least 150, at least 200, at least 250, at least 500, atleast 1000, at least 2500, or at least 5000 consecutive nucleotides ofSEQ ID NO: 87, 91, 95, 98, 105, 109, 113, 117, 122, 126, 130 or 137, ora sequence complementary thereto.

Embodiment 189. A recombinant DNA donor template comprising two homologyarms including a first homology arm and a second homology arm, whereinthe first homology arm comprises a sequence that is at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 99% or 100% complementary to at least20, at least 25, at least 30, at least 35, at least 40, at least 45, atleast 50, at least 60, at least 70, at least 80, at least 90, at least100, at least 150, at least 200, at least 250, at least 500, at least1000, at least 2500, or at least 5000 consecutive nucleotides of a firstflanking DNA sequence, wherein the second homology arm comprises asequence that is at least 70%, at least 75%, at least 80%, at least 85%,at least 90%, at least 95%, at least 96%, at least 97%, at least 99% or100% complementary to at least 20, at least 25, at least 30, at least35, at least 40, at least 45, at least 50, at least 60, at least 70, atleast 80, at least 90, at least 100, at least 150, at least 200, atleast 250, at least 500, at least 1000, at least 2500, or at least 5000consecutive nucleotides of a second flanking DNA sequence, and whereinthe first flanking DNA sequence and the second flanking DNA sequence aregenomic sequences at or near the genomic locus of an endogenous GAoxidase gene of a corn or cereal plant.

Embodiment 190. The recombinant DNA donor template of Embodiment 189,further comprising an insertion sequence located between the firsthomology arm and the second homology arm.

Embodiment 191. The recombinant DNA donor template of Embodiment 189,wherein the insertion sequence comprises at least 1, at least 2, atleast 3, at least 4, at least 5, at least 6, at least 7, at least 8, atleast 9, at least 10, at least 20, at least 30, at least 40, at least50, at least 60, at least 70, at least 80, at least 90, at least 100, atleast 200, at least 300, at least 400, at least 500, at least 750, atleast 1000, at least 2500, or at least 5000 nucleotides.

Embodiment 192. The recombinant DNA donor template of Embodiment 189,wherein each homology arm is at least 70%, at least 75%, at least 80%,at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 99% or 100% identical or complementary to at least 20, at least25, at least 30, at least 35, at least 40, at least 45, at least 50, atleast 60, at least 70, at least 80, at least 90, at least 100, at least150, at least 200, at least 250, at least 500, at least 1000, at least2500, or at least 5000 consecutive nucleotides of SEQ ID NO: 34, 35 or38, or a sequence complementary thereto.

Embodiment 193. The recombinant DNA donor template of Embodiment 189,wherein each homology arm is at least 70%, at least 75%, at least 80%,at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 99% or 100% identical or complementary to at least 20, at least25, at least 30, at least 35, at least 40, at least 45, at least 50, atleast 60, at least 70, at least 80, at least 90, at least 100, at least150, at least 200, at least 250, at least 500, at least 1000, at least2500, or at least 5000 consecutive nucleotides of SEQ ID NO: 87, 91, 95,98, 105, 109, 113, 117, 122, 126, 130 or 137, or a sequencecomplementary thereto.

Embodiment 194. The recombinant DNA donor template of Embodiment 189,wherein one or more nucleotides present in the genome of the monocot orcereal plant between the first flanking DNA sequence and the secondflanking DNA sequence are absent in the recombinant DNA donor templatemolecule between the first homology arm and the second homology arm.

Embodiment 195. The recombinant DNA donor template of Embodiment 194,wherein at least 1, at least 2, at least 3, at least 4, at least 5, atleast 6, at least 7, at least 8, at least 9, at least 10, at least 20,at least 30, at least 40, at least 50, at least 60, at least 70, atleast 80, at least 90, at least 100, at least 200, at least 300, atleast 400, at least 500, at least 750, at least 1000, at least 2500, orat least 5000 nucleotides present in the genome of the monocot or cerealplant between the first and second flanking DNA sequences are absent inthe recombinant DNA donor template molecule between the first and secondhomology arms.

Embodiment 196. A DNA molecule or vector comprising the recombinant DNAdonor template of Embodiment 189.

Embodiment 197. A bacterial or host cell comprising the recombinant DNAdonor template of Embodiment 189.

Embodiment 198. A corn or cereal plant, plant part or plant cellcomprising the recombinant DNA construct of Embodiment 189.

Embodiment 199. An engineered site-specific nuclease that binds to atarget site at or near the genomic locus of an endogenous GA oxidasegene of a corn or cereal plant and causes a double-strand break or nickat the target site.

Embodiment 200. The engineered site-specific nuclease of Embodiment 199,wherein the site-specific nuclease is a meganuclease or homingendonuclease.

Embodiment 201. The engineered site-specific nuclease of Embodiment 200,wherein the engineered meganuclease or homing endonuclease comprises ascaffold or base enzyme selected from the group consisting of I-CreI,I-CeuI, I-MsoI, I-SceI, I-AniI, and I-DmoI.

Embodiment 202. The engineered site-specific nuclease of Embodiment 199,wherein the site-specific nuclease is a zinc finger nuclease (ZFN)comprising a DNA binding domain and a cleavage domain.

Embodiment 203. The engineered zinc finger nuclease of Embodiment 202,wherein the cleavage domain is a FokI nuclease domain.

Embodiment 204. The engineered site-specific nuclease of Embodiment 199,wherein the site-specific nuclease is a transcription activator-likeeffector nuclease (TALEN) comprising a DNA binding domain and a cleavagedomain.

Embodiment 205. The engineered TALEN of Embodiment 204, wherein thecleavage domain is selected from the group consisting of a PvuIInuclease domain, a MutH nuclease domain, a Tell nuclease domain, a FokInuclease domain, an AlwI nuclease domain, a MlyI nuclease domain, a SbfInuclease domain, a SdaI nuclease domain, a StsI nuclease domain, aCleDORF nuclease domain, a Clo051 nuclease domain, and a Pept071nuclease domain.

Embodiment 206. The engineered site-specific nuclease of Embodiment 199,wherein the target site bound by the site-specific nuclease is at least80%, at least 85%, at least 90%, at least 95%, at least 96%, at least97%, at least 99% or 100% identical or complementary to at least 20, atleast 25, at least 30, at least 35, at least 40, at least 45, at least50, at least 60, at least 70, at least 80, at least 90, at least 100, atleast 150, at least 200, at least 250, at least 500, at least 1000, atleast 2500, or at least 5000 consecutive nucleotides of SEQ ID NO: 34,35 or 38, or a sequence complementary thereto.

Embodiment 207. The engineered site-specific nuclease of Embodiment 199,wherein the target site bound by the site-specific nuclease is at least80%, at least 85%, at least 90%, at least 95%, at least 96%, at least97%, at least 99% or 100% identical or complementary to at least 20, atleast 25, at least 30, at least 35, at least 40, at least 45, at least50, at least 60, at least 70, at least 80, at least 90, at least 100, atleast 150, at least 200, at least 250, at least 500, at least 1000, atleast 2500, or at least 5000 consecutive nucleotides of SEQ ID NO: 87,91, 95, 98, 105, 109, 113, 117, 122, 126, 130 or 137, or a sequencecomplementary thereto.

Embodiment 208. A recombinant DNA construct comprising a transgeneencoding a site-specific nuclease, wherein the site-specific nucleasebinds to a target site at or near the genomic locus of an endogenous GAoxidase gene of a monocot or cereal plant and causes a double-strandbreak or nick at the target site.

Embodiment 209. The recombinant DNA construct of Embodiment 208, whereinthe transgene is operably linked to a plant-expressible promoter.

Embodiment 210. The recombinant DNA construct of Embodiment 208, whereinthe site-specific nuclease is a meganuclease or homing endonuclease, azinc finger nuclease, or a transcription activator-like effectornuclease (TALEN).

Embodiment 211. A DNA molecule or vector comprising the recombinant DNAconstruct of Embodiment 208.

Embodiment 212. A bacterial or host cell comprising the recombinant DNAconstruct of Embodiment 208.

Embodiment 213. A corn or cereal plant, plant part or plant cellcomprising the recombinant DNA construct of Embodiment 208.

Embodiment 214. A recombinant DNA donor template comprising at least onehomology arm and an insertion sequence, wherein the at least onehomology arm is at least 70%, at least 75%, at least 80%, at least 85%,at least 90%, at least 95%, at least 96%, at least 97%, at least 99% or100% complementary to at least 20, at least 25, at least 30, at least35, at least 40, at least 45, at least 50, at least 60, at least 70, atleast 80, at least 90, at least 100, at least 150, at least 200, atleast 250, at least 500, at least 1000, at least 2500, or at least 5000consecutive nucleotides of a genomic DNA sequence of a corn or cerealplant, and wherein the insertion sequence comprises a recombinant DNAconstruct comprising a transcribable DNA sequence encoding a non-codingRNA molecule, wherein the non-coding RNA molecule targets forsuppression one or more endogenous GA20 or GA3 oxidase genes in amonocot or cereal plant or plant cell, and wherein the transcribable DNAsequence is operably linked to a plant-expressible promoter.

Embodiment 215. The recombinant DNA donor template of Embodiment 214,wherein the at least one homology arm comprises two homology armsincluding a first homology arm and a second homology arm, wherein thefirst homology arm comprises a sequence that is at least 70%, at least75%, at least 80%, at least 85%, at least 90%, at least 95%, at least96%, at least 97%, at least 99% or 100% complementary to at least 20, atleast 25, at least 30, at least 35, at least 40, at least 45, at least50, at least 60, at least 70, at least 80, at least 90, at least 100, atleast 150, at least 200, at least 250, at least 500, at least 1000, atleast 2500, or at least 5000 consecutive nucleotides of a first flankingDNA sequence, and the second homology arm comprises a sequence that isat least 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, at least 96%, at least 97%, at least 99% or 100%complementary to at least 20, at least 25, at least 30, at least 35, atleast 40, at least 45, at least 50, at least 60, at least 70, at least80, at least 90, at least 100, at least 150, at least 200, at least 250,at least 500, at least 1000, at least 2500, or at least 5000 consecutivenucleotides of a second flanking DNA sequence, wherein the firstflanking DNA sequence and the second flanking DNA sequence are genomicsequences at or near the same genomic locus of a monocot or cerealplant, and wherein the insertion sequence is located between the firsthomology arm and the second homology arm and comprises a recombinant DNAconstruct comprising a transcribable DNA sequence encoding a non-codingRNA molecule.

Embodiment 216. The recombinant DNA donor template of Embodiment 215,wherein the transcribable DNA sequence is operably linked to aplant-expressible promoter.

Embodiment 217. The recombinant DNA donor template of Embodiment 215,wherein the non-coding RNA molecule comprises a sequence that is atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, at least 99.5%, or 100% complementary to at least 15, atleast 16, at least 17, at least 18, at least 19, at least 20, at least21, at least 22, at least 23, at least 24, at least 25, at least 26, orat least 27 consecutive nucleotides of a mRNA molecule encoding a GAoxidase protein that is at least 80%, at least 85%, at least 90%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, atleast 99.5%, or 100% identical to SEQ ID NO: 9, 12, 15, 30 or 33.

Embodiment 218. The recombinant DNA donor template of Embodiment 215,wherein the non-coding RNA molecule comprises a sequence that is atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, at least 99.5%, or 100% complementary to at least 15, atleast 16, at least 17, at least 18, at least 19, at least 20, at least21, at least 22, at least 23, at least 24, at least 25, at least 26, orat least 27 consecutive nucleotides of a mRNA molecule encoding a GAoxidase protein that is at least 80%, at least 85%, at least 90%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, atleast 99.5%, or 100% identical to SEQ ID NO: 86, 90, 94, 97, 101, 104,108, 112, 116, 118, 121, 125, 129, 133, or 136.

Embodiment 219. A composition comprising the recombinant DNA donortemplate of Embodiment 214.

Embodiment 220. A bacterial or host cell comprising the recombinant DNAdonor template of Embodiment 214.

Embodiment 221. A transgenic corn or cereal plant, plant part or plantcell comprising the insertion sequence of the recombinant DNA donortemplate of Embodiment 214.

Embodiment 222. The transgenic corn or cereal plant of Embodiment 214,wherein the transgenic plant has one or more of the following traitsrelative to a control plant: shorter plant height, increased stalk/stemdiameter, improved lodging resistance, deeper roots, increased leafarea, earlier canopy closure, higher stomatal conductance, lower earheight, increased foliar water content, improved drought tolerance,improved nitrogen use efficiency, reduced anthocyanin content and areain leaves under normal or nitrogen or water limiting stress conditions,increased ear weight, increased harvest index, increased yield,increased seed number, increased seed weight, and/or increasedprolificacy.

Embodiment 223. The transgenic corn or cereal plant of Embodiment 222,wherein the transgenic plant has a shorter plant height and/or improvedlodging resistance.

Embodiment 224. The transgenic corn or cereal plant of Embodiments 222,wherein the height of the transgenic plant is at least 10%, at least20%, at least 25%, at least 30%, at least 35%, or at least 40% shorterthan a control plant.

Embodiment 225. The transgenic corn or cereal plant of Embodiments 222,wherein the level of one or more active GAs in at least one internodetissue of the stem or stalk of the transgenic plant is lower than thesame internode tissue of a control plant.

Embodiment 226. A method for producing a transgenic corn or cerealplant, comprising: (a) transforming at least one cell of an explant withthe recombinant DNA donor template of Embodiment 215, and (b)regenerating or developing the transgenic corn or cereal plant from thetransformed explant, wherein the transgenic corn or cereal plantcomprises the insertion sequence of the recombinant DNA donor template.

Embodiment 227. The method of Embodiment 226, wherein the monocot orcereal plant is transformed via Agrobacterium mediated transformation orparticle bombardment.

Embodiment 228. A method for producing a corn or cereal plant having agenomic edit at or near an endogenous GA oxidase gene, comprising: (a)introducing into at least one cell of an explant of the corn or cerealplant a site-specific nuclease or a recombinant DNA molecule comprisinga transgene encoding the site-specific nuclease, wherein thesite-specific nuclease binds to a target site at or near the genomiclocus of the endogenous GA oxidase gene and causes a double-strand breakor nick at the target site, and (b) regenerating or developing an editedcorn or cereal plant from the at least one explant cell comprising thegenomic edit at or near the endogenous GA oxidase gene of the editedcorn or cereal plant.

Embodiment 229. The method of Embodiment 228, wherein the introducingstep (a) further comprises introducing a DNA donor template comprisingat least one homology sequence or homology arm, wherein the at least onehomology sequence or homology arm is at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 99% or 100% complementary to at least 20, at least25, at least 30, at least 35, at least 40, at least 45, at least 50, atleast 60, at least 70, at least 80, at least 90, at least 100, at least150, at least 200, at least 250, at least 500, at least 1000, at least2500, or at least 5000 consecutive nucleotides of a target DNA sequence,wherein the target DNA sequence is a genomic sequence at or near thegenomic locus of the endogenous GA oxidase gene of the corn or cerealplant.

Embodiment 230. The method of Embodiment 228, further comprising: (c)selecting the edited corn or cereal plant.

Embodiment 231. The method of Embodiment 230, wherein the selecting step(c) comprises determining if the endogenous GA oxidase gene locus wasedited using a molecular assay.

Embodiment 232. The method of Embodiment 230, wherein the selecting step(c) comprises determining if the endogenous GA oxidase gene was editedby observing a plant phenotype.

Embodiment 233. The method of Embodiment 231, wherein the plantphenotype is a decrease in plant height relative to a control plant.

Embodiment 234. The method of Embodiment 228, wherein the introducingstep (a) creates at least one mutation at or near the genomic locus ofthe endogenous GA oxidase gene, and wherein the mutation comprises asubstitution, deletion, insertion, or inversion of one or morenucleotides relative to the genomic DNA sequence of a control plant.

Embodiment 235. A modified corn plant having a plant height of less than2000 mm, less than 1950 mm, less than 1900 mm, less than 1850 mm, lessthan 1800 mm, less than 1750 mm, less than 1700 mm, less than 1650 mm,less than 1600 mm, less than 1550 mm, less than 1500 mm, less than 1450mm, less than 1400 mm, less than 1350 mm, less than 1300 mm, less than1250 mm, less than 1200 mm, less than 1150 mm, less than 1100 mm, lessthan 1050 mm, or less than 1000 mm, and either (i) an average stem orstalk diameter of greater than 18 mm, greater than 18.5 mm, greater than19 mm, greater than 19.5 mm, greater than 20 mm, greater than 20.5 mm,greater than 21 mm, greater than 21.5 mm, or greater than 22 mm, (ii)improved lodging resistance relative to a wild type control plant, or(iii) improved drought tolerance relative to a wild type control plant.

Embodiment 236. The modified corn plant of Embodiment 235, wherein thecorn plant has one or more of the following traits relative to a wildtype control plant: increased stalk/stem diameter, improved lodgingresistance, deeper roots, increased leaf area, earlier canopy closure,higher stomatal conductance, lower ear height, increased foliar watercontent, improved drought tolerance, improved nitrogen use efficiency,reduced anthocyanin content and area in leaves under normal or nitrogenor water limiting stress conditions, increased ear weight, increasedharvest index, increased yield, increased seed number, increased seedweight, and/or increased prolificacy.

Embodiment 237. The modified corn plant of Embodiment 235, wherein thelevel of one or more active GAs in at least one internode tissue of thestem or stalk of the corn plant is lower than the same internode tissueof a wild type control plant.

Embodiment 238. A modified cereal plant having a reduced plant heightrelative to a wild type control plant, and (i) an increased stem orstalk diameter relative to a wild type control plant, (ii) improvedlodging resistance relative to a wild type control plant, or (iii)improved drought tolerance relative to a wild type control plant.

Embodiment 239. The modified cereal plant of Embodiment 238, wherein thelevel of one or more active GAs in the stem or stalk of the cereal plantis lower than in a wild type control plant.

EXAMPLES Example 1. Reduced Plant Height in Inbred Corn Lines AcrossTransformation Events for the GA20 Oxidase Suppression Element

An inbred corn plant line was transformed via Agrobacterium mediatedtransformation with a transformation vector having an expressionconstruct comprising a transcribable DNA sequence with a sequence (SEQID NO: 39) encoding a targeting sequence (SEQ ID NO: 40) of a miRNAunder the control of a rice tungro bacilliform virus (RTBV) promoter(SEQ ID NO: 65) that is known to cause expression in vascular tissues ofplants. The miRNA encoded by the construct comprises a RNA sequence thattargets the GA20 oxidase_3 and GA20 oxidase_5 genes in corn plants forsuppression. Several transformation events were generated with thisconstruct, and these transformants were tested in the greenhouse todetermine if they had reduced plant height relative to non-transgenicwild type control plants. As can be seen in FIG. 1 , a significantreduction in plant height was consistently observed in transgenic plantsexpressing the suppression construct across several transformationevents (see Events 1-8) relative to wild type (WT) control plants. Plantheight for each of the transformation events was calculated as anaverage among approximately 10 plants for each event and compared to theaverage height for control plants. Standard errors were calculated foreach event and the control plants, which are represented as error barsin FIG. 1 . Furthermore, ear development in each of these transformantsappeared normal.

As can be seen from the results of this experiment, average plant heightin plants expressing the miRNA targeting the GA20 oxidase_3 and GA20oxidase_5 genes for suppression had consistently reduced plant heightsof up to 35% relative to control plants across multiple events. Thisdata supports the conclusion that the effects seen with this suppressionconstruct are not due to insertion of the construct at any one locuswithin the plant genome.

This data further indicates that expression of this GA20 oxidasesuppression construct using the RTBV vascular promoter is effective atcausing these plant height phenotypes. In addition, early data in R0corn plants constitutively expressing the same GA20 oxidase suppressionconstruct under the control of different constitutive promoters alsoproduce short stature plants (see Example 15 below). Thus, expression ofthe targeted GA20 oxidase suppression construct may be effective atreducing plant height and providing the other beneficial anti-lodgingand yield-related traits described herein given that differentexpression patterns including vascular and constitutive expressionprovide similar plant height phenotypes without apparent off-types inthe ear.

Example 2. Reduced Plant Height in Hybrid Corn Plants Expressing theGA20 Oxidase Suppression Element

Hybrid corn plants carrying the GA20 oxidase suppression constructdescribed in Example 1 also showed reduced plant height relative to wildtype control plants when grown under field conditions. Average plantheight of transgenic hybrid corn plants expressing the GA20 oxidasesuppression element in 10 microplots was calculated and compared toaverage plant height of (non-transgenic) wild type control hybrid cornplants in 32 microplots. Each microplot for the transgenic andnon-transgenic control included approximately 6 plants, although theactual number of plants per plot may vary depending on the number ofplants that germinate and develop into plants having ears. As can beseen in FIG. 2A, a significant reduction in average plant height wasobserved in transgenic hybrid plants expressing the suppressionconstruct (SUP-GA20ox hybrid), relative to wild type hybrid corn plants(Control). Standard errors were calculated for the transgenic hybrid andcontrol plants, which are represented as error bars in FIG. 2A. An imageof a hybrid control plant (left) next to a transgenic hybrid plantexpressing the GA20 oxidase suppression element (right) is further shownin FIG. 2B.

In this experiment, average plant height of field grown hybrid cornplants expressing the miRNA targeting the GA20 oxidase_3 and GA20oxidase_5 genes was reduced by about 40% relative to wild type hybridcontrol plants. This data shows that the plant height phenotype ispresent in hybrid corn plants in addition to inbred lines. However,overall biomass in this experiment appeared neutral in the semi-dwarfcorn plants compared to controls.

Example 3. Increased Stem Diameter in Hybrid Corn Plants Expressing theGA20 Oxidase Suppression Element

Hybrid corn plants carrying the GA20 oxidase suppression constructdescribed in Example 1 also showed increased stem diameter relative towild type control plants when grown under field conditions. Stemdiameter was measured on the second internode below the primary ear.Average stem diameter of transgenic hybrid corn plants expressing theGA20 oxidase suppression element in 8 microplots was calculated andcompared to the average stem diameter of (non-transgenic) wild typecontrol hybrid corn plants in 8 microplots. Each microplot includedapproximately 6 plants. As can be seen in FIG. 3A, a significantincrease in average stem diameter was observed in transgenic hybridplants expressing the suppression construct (SUP-GA20ox hybrid),relative to wild type hybrid corn plants (Control). Standard errors werecalculated for the transgenic hybrid and control plants, which arerepresented as error bars in FIG. 3A. An image of the cross-section of astalk from a hybrid control plant (Control; left) is shown next to thecross-section of a stalk from a transgenic hybrid plant expressing theGA20 oxidase suppression element (SUP GA20ox; right) is further shown inFIG. 3B.

In this experiment, average stem diameter of field grown hybrid cornplants expressing the miRNA targeting the GA20 oxidase_3 and GA20oxidase_5 genes was increased about 13% relative to wild type hybridcontrol plants. This data shows that hybrid corn plants expressing theGA20 oxidase miRNA may have thicker stalks in addition to the reducedplant height phenotype.

Example 4. Hybrid Corn Plants Expressing the GA20 Oxidase SuppressionElement had an Increase in Fresh Ear Weight

Hybrid corn plants carrying the GA20 oxidase suppression constructdescribed in Example 1 also showed an increase in fresh ear weightrelative to wild type control plants when grown under field conditions.Average fresh ear weight per plot of transgenic hybrid corn plantsexpressing the GA20 oxidase suppression element in 24 microplots wascalculated and compared to the average fresh ear weight of(non-transgenic) hybrid corn control plants in 8 microplots. Again, eachmicroplot included about 6 plants. As can be seen in FIG. 4 , anincrease in average fresh ear weight per plot was observed in transgenichybrid plants expressing the suppression construct (SUP-GA20ox hybrid),relative to wild type hybrid corn plants (Control), and ear and kerneldevelopment appeared normal. Standard deviations for this experimentwere calculated for the transgenic hybrid and control plants, which arerepresented as error bars in FIG. 4 . As shown in FIG. 5 , similarresults were obtained at another field testing site that alsoexperienced wind damage.

In this experiment, average fresh ear weight of field grown hybrid cornplants expressing the miRNA targeting the GA20 oxidase_3 and GA20oxidase_5 genes was increased relative to wild type hybrid controlplants, indicating that these transgenic plants may further haveimproved yield-related traits. However, these results are based onobservational data without a large-scale statistical comparison tocontrols, and yield performance should be tested under broad acreconditions.

Example 5. Hybrid Corn Plants Expressing the GA20 Oxidase SuppressionElement Displayed Increased Resistance to Lodging

At a field testing location, wind damage to pre-flowering hybrid cornplants demonstrated an increased lodging resistance with plantsexpressing the GA20 oxidase suppression construct described in Example1, relative to wild type hybrid control plants. While the wild type(non-transgenic) hybrid control plants were visually lodged in responseto this high wind event, transgenic hybrid corn plants expressing theGA20 oxidase suppression element in a neighboring field locationresisted lodging damage. To evaluate the effects of lodging resistanceby hybrid corn plants expressing the GA20 oxidase suppression construct,average fresh ear weights per plot of transgenic GA20oxidase-suppressing hybrid corn plants across two field trial locationsexperiencing the lodging damage, were compared to average fresh earweights of wild type hybrid control plants. Data collected from thesetwo trials indicated that the hybrid control plants had average freshear weights that were reduced by about 57% and 81%, respectively in thetwo trials, relative to hybrid plants expressing the GA20 oxidasesuppression construct.

The visual observation that transgenic GA20 oxidase-suppressing hybridcorn plants had increased lodging resistance than non-transgenic controlplants, along with the increase in average fresh ear weight in thesetrials with the transgenic GA20 oxidase-suppressing plants, indicatethat increased lodging resistance may translate into an increase inaverage fresh ear weight. Thus, increased lodging resistance in GA20oxidase-suppressing plants may further increase the yieldpotential/stability of these transgenic corn plants by resisting theeffects of lodging during severe weather events.

Example 6. Hybrid Corn Plants Expressing the GA20 Oxidase SuppressionElement had an Increase in Harvest Index

Hybrid corn plants carrying the GA20 oxidase suppression constructdescribed in Example 1 further showed an increase in harvest indexrelative to wild type control plants when grown under field conditions.The harvest index of transgenic hybrid corn plants expressing the GA20oxidase suppression element was determined from plants grown in 8microplots and compared to non-transgenic hybrid corn control plants.Each microplot included approximately 6 plants. As can be seen in FIG. 6, a significant increase in harvest index was observed in the transgenichybrid plants expressing the suppression construct (SUP-GA20ox hybrid),relative to wild type hybrid corn plants (Control). Standard errors werecalculated for the transgenic hybrid and control plants, which arerepresented as error bars in FIG. 6 .

In this experiment, the harvest index of field grown hybrid corn plantsexpressing the miRNA targeting the GA20 oxidase_3 and GA20 oxidase_5genes was increased about 11% relative to wild type hybrid controlplants. This increase in harvest index was further associated with areduction in stover weight as compared to wild type control plants, butno difference in total dry biomass weight was observed in the transgenicplants.

Example 7. Hybrid Corn Plants Expressing the GA20 Oxidase SuppressionElement had an Increase in Average Grain Yield Estimate

The average grain yield estimate for hybrid corn plants expressing theGA20 oxidase suppression element (identified in Example 1) wascalculated from 16 microplots in the field (with approximately 6 plantsper plot). The calculated average grain yield estimate for thesetransgenic hybrid corn plants suppressing GA20 oxidase was increased byabout 15% over corn hybrid control plants (FIG. 7 ). Grain yieldestimate is a metric that provides a general estimation of expectedyield based on the ear trait metrics. Grain yield estimate is derivedfrom hand harvested ears on small plots, and units are kg/ha (instead ofbu/ac). Grain yield estimate (kg/ha) is calculated by the formula(Kernel number per unit area (kernels/m²)×Single Kernel Weight(mg)×15.5%/100).

Example 8. Hybrid Corn Plants Expressing the GA20 Oxidase SuppressionElement had an Increase in Average Prolificacy Score

Hybrid corn plants expressing the GA20 oxidase suppression element(identified in Example 1) was also shown in a microplot experiment tohave increased prolificacy and secondary ears as compared tonon-transgenic hybrid control plants. The prolificacy score wasdetermined from 10 microplots of the transgenic hybrid corn plants inthe field (with approximately 6 plants per plot). As shown in FIG. 8 ,the average prolificacy score of transgenic hybrid corn plantssuppressing the GA 20 oxidase was 3, whereas the average prolificacyscore of control plants was 1. To determine the prolificacy score,plants were assayed for the development of secondary ears at the R1stage of development. Plants were rated on the following scale: 1=Littleor no secondary ear formation; 2=Silks are prominent on the secondaryear; 3=Developed secondary ear emerged from the ear leaf sheath; and4=Good secondary ear development similar to the primary ear.End-of-season harvest further indicated at least some secondary earswere productive with normally developed kernels.

Example 9. Broad-Acre Yield and Trait Trials in the Field with HybridCorn Plants Transformed with the GA20 Oxidase Suppression Construct

The GA20 oxidase suppression construct described in Example 1 wastransformed into a female commercial corn inbred line, and a number oftransformation events were created. The transformed plants were grownand self-crossed to bulk up sufficient seeds, and then crossed tovarious male commercial corn inbred lines to produce hybrid corn plants.Each distinct male inbred line used to produce the male-female hybrid iscalled a tester. The hybrid corn plants with different testers were thengrown on broad acres in the field according to standard agronomicpractice (SAP). The planting density for SAP was 34,000 plants per acreswith 30″ row spacing.

For yield trials, four different transformation events expressing theGA20 oxidase suppression construct were crossed to 2 differentcommercial tester lines. The hybrid corn plants were then tested in 16geographic locations across 6 US Midwest states. Yield of transgenichybrid corn plants across these locations was calculated and compared tothe yield of non-transgenic hybrid corn control plants. Table 4 providesthe yield difference in bushels/acre between the transgenic hybrid cornplants for each event as compared to a non-transgenic control. Anegative number indicates a yield decrease. Yield differences with astatistical p-value of less than 0.2 are indicated in Table 4 with boldand italic font. This notation is also used to indicate statisticalsignificance for the remaining tables in these Examples, unlessotherwise noted. As shown in Table 4 under the SAP heading, asignificant increase in yield was observed in transgenic hybrid cornplants expressing the suppression construct (transgenic plants) underSAP conditions, relative to wild type hybrid corn plants (Control). Thesignificant increase in yield was observed across 4 transgenic events,and 2 tester lines.

A comparable broad-acre yield trial was conducted under high density(HD) planting conditions with 42,000 plants per acre and 30″ rowspacing, and compared to standard agronomic practice (SAP) density. Thedifferences in yield under HD conditions are provided in Table 4 underthe HD heading. Mixed results were obtained under these high densityconditions with yield varying across events and testers. However, anincrease in yield was observed for two events with one of the twotesters, and the possibility remains for higher yield across a greaternumber of germplasms under different high density conditions.

TABLE 4 Broad-acre yield difference between transgenic plants andcontrol, under SAP and HD SAP HD Across Across Tester-1 Tester-2 TestersTester-1 Tester-2 Testers Across Events 3.7 3.9 4 3.5

−3.9 Event-1

2.7

−4.3 1.3 Event-2 3.2

−5.1

Event-3 2.3 1.8 2

−0.7 Event-4 1.7 4.6 3.4 3.1

Trait trials were also conducted in the field to measure a number ofdevelopmental and reproductive traits. These trials were conducted undernormal density (SAP) as described above and ultra high density (UHD)planting conditions of 54,000 plants per acre with 20″ row spacing. Thetrials were conducted in hybrid corn plants with 7 transformation eventsand 3 testers, and the data for each tester was pooled over the 7events.

Table 5 summarizes the trait trial results in hybrid corn plants. Themeasurement is either a percent difference, or a difference of days ornumber of leaves, between the transgenic plants and the control. Whereappropriate, the development stage, such as R3, etc., at which themeasurement was taken, is indicated in parenthesis under the column“Trait Name”. Pollen shedding is measured in terms of the number of daysfrom germination to 50% of plants shedding pollen. Silking emergence ismeasured in terms of the number of days from germination to 50% ofplants silking. Pollen-silk interval is a measure of the number of daysfrom 50% of plants shedding pollen to silking. Stalk strength is ameasure of the amount of force at which the stalk segment breakslaterally, using a stalk breaker instrument. Leaf area index (LAI) is adimensionless quantity that characterizes the extent of the plantcanopy, defined as the one-sided green leaf area per unit ground surfacearea within a broadleaf canopy space.

TABLE 5 Trait differences between transgenic and control plants underSAP and UHD. 30″ SAP 20″ UHD Measurement Trait Name Tester-1 Tester-2Tester-3 Tester-1 Tester-2 Tester-3 % Delta Plant height (R3)

Plant height below 6 ft

Ear height (R3)

Ear height above 18

inches % Delta Internode length(ear

minus 2) (R3) Internode length(ear

minus 4) (R3) Stalk Diameter (2 4.4 5.8 4.5

nodes below ear) (R3) Stalk Diameter (4 3.9 −1.6 1

nodes below ear) (R3) Stalk strength 2nd 10.2 0.1 0.7

N/A node below ear (R5) Stalk strength 4th node −13.6

−11.5

N/A below ear (R5) Days Pollen-silk interval

Pollen shedding

Silking emergence

−0.25

Number Green leaf # (R4)

Green leaf # (R5)

Green leaf # (7 days −0.5

−0.3

−0.3

after R5) Green leaf # (14 days

−0.4 −0.2

−0.3

after R5) % Delta Leaf area index (V6)

−16.6 Leaf area index (V8)

−1.4 15.4

Leaf area index (V10) 10.7 2 8.5

−7.7 Leaf area index (V12) 2.3 −5.4 3.6

As shown in Table 5, a significant decrease in plant height, ear height,and internode length was observed in transgenic plants relative to thecontrol. The transgenic plants consistently exhibited plant heightsbelow 6 feet, and ear heights above 18 inches, allowing harvesting bycombine without modification to the machinery. In this experiment,increased stalk diameter was observed particularly under higher densityplanting conditions.

Table 6 summarizes the ear trait trial results for hybrid corn. Thetrials were conducted in hybrid corn plants with 7 transformation eventsand 3 testers, and the data for each tester was pooled over the 7events. The measurements are the percent delta difference between thetransgenic plants and the control. Where appropriate, the developmentstage, such as R3, etc., at which the measurement was taken, isindicated in parenthesis under the column “Trait Name”. Ear area is ameasure of the plot average size of an ear in terms of area from a2-dimensional view taken by imaging the ear, including kernels and void.Ear diameter is a measures the plot average of the ear diameter measuredas the maximal “wide” axis of the ear over its widest section. Earlength is a measure of the plot average of the length of ear measuredfrom the tip of the ear in a straight line to the base of the ear node.Ear tip void_pct is a measure of the plot average of the area percentageof void at the top 30% area of the ear, from a 2-dimensional view takenby imaging the ear, including kernels and void. Ear void measures theplot average of the area percentage of void on an ear, from a2-dimensional view, is measured by imaging the ear, including kernelsand void. Grain yield estimate is defined in Example 7. Kernels per unitarea is measured as the plot average of the number of kernels per unitarea of the field. Ears were collected from a set row length, typicallyone meter, and shelled and combined to count the kernels, and the countwas then converted to the total kernels per unit area of the field.Single kernel weight measures the plot average of weight per kernel. Itis calculated as the ratio of (sample kernel weight adjusted to 15.5%moisture)/(sample kernel number). Kernels per ear is a measure of theplot average of the number of kernels per ear. It is calculated as(total kernel weight/(Single Kernel Weight*total ear count), where totalkernel weight and total ear count are measured from ear samples over anarea between 0.19 to 10 square meters.

TABLE 6 Ear trait differences between transgenic and control plants,under SAP and UHD. 30″ SAP 20″ UHD Trait Name Tester-1 Tester-2 Tester-3Tester-1 Tester-2 Tester-3 Ear area (R6) (cm²)

Ear diameter (R6) (mm)

−0.7

−1.7 1.3 −1.8 Ear length (R6) (cm)

Ear tip void_pct (R6) (%) −9.1 −1.1 7.7 −5.8 24 11.1 Ear void (R6) (%)−3.3 1.9 9.5 −6.7 10 16.4 Grain yield estimate (R6) 2.8 −4.6 −5.0 0.219.2 0.9 (kg/hectare) Kernels per unit area (R6) −0.7 −9.8 −6.7

9 (kernels/m²) Kernels per ear (R6) (count) −3.2 0.5 −3.5

6.5 Single kernel weight (R6) 1.8 5.1 1.1

−7.5 (mg)

As shown in Table 6, there was a significant increase in ear area andear length observed in these experiments for the transgenic plants ascompared to the control. There was also a noticeable decrease in the eardiameter. In this experiment, the grain yield estimate was mostlyneutral between transgenic plants and the control.

Additional data was collected in the field at standard density across 8events crossed to one tester showing a reduction in plant height, earheight, and internode length, and an increase in stem diameter andharvest index, as compared to a control (data not shown). Plant heightswere measured from the ground to the uppermost ligulated leaf at R3stage. Ear heights were measured from the ground to the ear node at R3stage. Stalk diameters were measured at the middle of the stalkinternode 2 nodes below the ear, unless otherwise indicated. These datademonstrated high penetrance of plant height and stalk traits acrossevents, although an increase in prolificacy (or the number of secondaryears) was not significant or pronounced in these studies.

In a separate experiment, plant height growth was measured from V11 toR1 stage and beyond. FIG. 9 shows the differences in plant heightbetween transgenic plants and the control over this time frame. Drawn onthe figure are dotted lines for 5-foot and 6-foot heights for reference.

Example 10. Transgenic Plants Exhibited Enhanced Traits Under Nitrogenand Water Stress Conditions in Controlled Environment Conditions

This example illustrates the enhanced water and nitrogen stress responseof transgenic corn plants having the GA20 oxidase suppression constructdescribed in Example 1 versus the control, in an automated greenhouse(AGH) or the field as indicated. The apparatus and the methods forautomated phenotypic assaying of plants in AGH are disclosed, forexample, in U.S. Patent Publication No. 2011/0135161, which isincorporated herein by reference in its entirety.

In the AGH setting, corn plants were tested under five differentconditions including non-stress, mild and moderate nitrogen deficit, andmild and moderate water deficit stress conditions. The corn plants weregrown under the stress-specific conditions shown in Table 7.

TABLE 7 Description of the five AGH growth conditions. Volumetric WaterNitrogen Condition Content (VWC) Concentration No stress 50% 8 mM WaterStress: mild 40% 8 mM Water Stress: moderate 35% 8 mM Nitrogen Stress:mild 50% 6 mM Nitrogen Stress: moderate 50% 4 mM

Water deficit is defined as a specific Volumetric Water Content (VWC)that is lower than the VWC of a non-stressed plant. For example, anon-stressed plant might be maintained at 50% VWC, and the VWC for awater-deficit assay might be defined between 35% to 40% VWC. Data werecollected using visible light and hyperspectral imaging as well asdirect measurement of pot weight and amount of water and nutrientapplied to individual plants on a daily basis. Nitrogen deficit isdefined (in part) as a specific mM concentration of nitrogen that islower than the nitrogen concentration of a non-stressed plant. Forexample, a non-stressed plant might be maintained at 8 mM nitrogen,while the nitrogen concentration applied in a nitrogen-deficit assaymight be maintained at a concentration between 4 to 6 mM.

Up to ten parameters were measured for each screen. The visible lightcolor imaging based measurements are: plant height, biomass, and canopyarea. Plant Height (PlntH) refers to the distance from the top of thepot to the highest point of the plant derived from a side image (mm).Biomass (Bmass) is defined as the estimated shoot fresh weight (g) ofthe plant obtained from images acquired from multiple angles of view.Canopy Area (Cnop) is defined as leaf area as seen in a top-down image(mm²). Anthocyanin score and area, chlorophyll score and concentration,and water content score were measured with hyperspectral imaging.Anthocyanin Score (AntS) is an estimate of anthocyanin in the leafcanopy obtained from a top-down hyperspectral image. Anthocyanin Area(AntA) is an estimate of anthocyanin in the stem obtained from aside-view hyperspectral image. Chlorophyll Score (ClrpS) and ChlorophyllConcentration (ClrpC) are both measurements of chlorophyll in the leafcanopy obtained from a top-down hyperspectral image, where ChlorophyllScore measures in relative units, and Chlorophyll Concentration ismeasured in parts per million (ppm) units. Foliar Water Content(FlrWtrCt) is a measurement of water in the leaf canopy obtained from atop-down hyperspectral image. Water Use Efficiency (WUE) is derived fromthe grams of plant biomass per liter of water added. Water Applied(WtrAp) is a direct measurement of water added to a pot (pot with nohole) during the course of an experiment. These physiological trialswere set up so that tested transgenic plants were compared to thecontrol. Transgenic plants of two transformation events were measured incomparison with the control. All data are in percent delta difference ofthe transgenic plant with respect to the control. Data point withstatistical p-value <0.1 were shown in bold italic font. Other datapoints have p-value >0.1.

Table 8 summarizes the AGH trait trial results as measured at 21 daysfrom planting in the vegetative stage, whereas Table 9 summarizes theAGH trait trial results as measured at 55 days from planting in thereproductive stage, in transgenic plants having one of two events of theGA20 oxidase suppression construct described in Example 1 relative tocontrol plants.

TABLE 8 Transgenic versus control plants in the greenhouse under normaland stress conditions, 21 days from planting. Event-1 Event-2 NoNitrogen stress Water stress Mn Nitrogen stress Water stress Trait Namestress Mild Moderate Mild Moderate stress Mild Moderate Mild ModeratePlant height

Biomass −0.06

−0.32

−1.77

−1.48 Canopy area 0.79

1.45

0.36

4.38 2.75 Foliar water

10.1

content Anthocyanin

area Anthocyanin −10.21 −14.5 −2.9

2.4

4.5 3.3

−2.5 score Chlorophyll 1.2 0.68 0.04

3.27

−2.03 −3.46 −4.14 2.05 concentration

TABLE 9 Transgenic versus control plants in the greenhouse under normaland stress conditions, 55 days from planting. Event-1 Event-2 NoNitrogen stress Water stress No Nitrogen stress Water stress Trait Namestress Mild Moderate Mild Moderate stress Mild Moderate Mild ModeratePlant height

N/A N/A

N/A N/A

Biomass

N/A N/A

N/A N/A

Ear weight

10.7

Stover weight

0

Harvest index

Water applied

N/A N/A

N/A N/A

−2.3 WUE

N/A N/A

N/A N/A

As shown in Table 8, in comparison with the control, transgenic plantsexhibited some enhanced traits related to stress resistance andmaintained other positive traits under stress conditions. The plantheight decreased significantly across all treatments and was notaffected by stress condition. Biomass and canopy area were neutral inno-stress condition but increased in more severe stress conditions. Thefoliar water content increased significantly in no-stress and stressconditions, indicating that the transgenic plants retained more water inleaf tissues. The anthocyanin area decreased significantly in no-stressand stress conditions, indicating there was no nitrogen deficiency inthe transgenic plants.

As shown in Table 9, in comparison with the control, transgenic plantsexhibited significant decrease in the trait areas of Water Applied, WUE,biomass and stover weight, indicating that the transgenic plants hadimproved water use efficiency, with plants of lower biomass requiringless water. Harvest index increased significantly under non-stress andstress conditions.

Example 11. Transgenic Plants Exhibited Increased Drought Tolerance,Stomatal Conductance, and Root Front Velocity at Reproductive Stages atBoth Standard and High Density in the Field

Direct observations were made of decreased leaf rolling in transgeniccorn plants having the GA20 oxidase suppression construct from Example 1under drought conditions in the field compared to control plants. Cornleaf rolling occurs when leaf water potential drops below a threshold ofapproximately −1.1 MPa. Stomatal conductance also decreases under waterstress. Stable oxygen isotope ratios (δ¹⁸O) were used as an index of thestomatal conductance, which is inversely proportional to stomatalconductance. A significant decrease of δ¹⁸O, and thus a significantincrease in the stomatal conductance, in transgenic plants over thecontrol was observed from ear leaf samples collected at R5 stage (seeFIG. 10 ). Data was taken from transgenic plants across twotransformation events and averaged across 10 testers with 2 reps pertester. Increased δ¹⁸O in the leaf of control plants indicates thatstomatal conductance was lower for the control. In conjunction with thereduced leaf rolling observed in the field, the significant increase ofstomatal conductance in leaves of transgenic plants from yield trials at15 out of 16 field locations indicates improved leaf water status duringlate vegetative growth for the transgenic plants.

Effective water uptake by the roots is an important factor in plantgrowth. To measure the developmental progress of rooting depth, Sentek®SOLO soil moisture capacitance probes were installed at V4 stage withinthe row between plants at one field location. Soil moisture was measuredon an hourly basis with capacitance sensors at depths of 10, 20, 30, 50,70, 90, 120, and 150 cm from the ground level. The depth of the rootingfront was inferred by the presence of diurnal patterns in soil moisturedepletion recorded by the sensors. Root activity was already present at10, 20, and 30 cm depth at the time of installation at V4 stage. Wedetected the first occurrence of soil moisture depletion at 50, 70, and90 cm depths. The soil at 120 and 150 cam depth was saturated throughoutthe growing season. While root growth may have reached these depths, wewere not able to detect root activity at these depths for thisexperiment due to the inability to detect soil moisture depletion in asaturated zone. FIG. 11 shows the time (days after planting) for thefrontal root of the plant to reach various depths on the Y axis. Lineswith circles are for plants at 30-inch row spacing and 34,000 plants peracre planting density, and lines with squares are for plants at 20-inchrow spacing, and 55,000 plants per acre planting density. Growth stagesare shown on the X axis.

As shown in FIG. 11 , root growth was similar in this experiment betweentransgenic and control plants up to V12, with roots reaching 50 and 70cm depth at about 30 and 36 days after planting, respectively. However,the transgenic plant roots reached 90 cm depth at or before R1 (i.e., atabout day 50 after planting), or about 20 days earlier than controlplant roots. The transgenic plants exhibited increased rooting frontvelocity after V11/V12 stage, which may lead to increased droughtavoidance during the critical period of plant development aroundflowering. This increase in rooting front velocity may allow thetransgenic plants to take advantage of deeper reserves of soil waterduring the critical period around R1 stage, possibly allowing droughteffects on flowering and pollination to be avoided, reduced orminimized. Improved pollination under drought conditions may likelyimprove kernel set and yield potential.

To complement the above field experiment with moisture sensors, rootfront velocity for transgenic corn plants having the GA20 oxidasesuppression construct from Example 1 (n=10) was measured in a root boxexperiment and compared to wild-type control plants (n=9). Plexiglassroot boxes (5 feet tall and six-by-eight inches in cross section; ½ inchwall thickness) were filled with a mix of #10 fieldsoil/vermiculite/perlite (1:1:1 ratio) and used for root visualizationfor each plant. Maximum rooting depth in each box was measured atregular intervals after planting (approximately every two days). In thisexperiment, median root front depth of transgenic plants wasconsistently greater or deeper than WT control plants starting at about21 days after planting (i.e., at about V4 stage) and continuing until atleast 34 days after planting when measurements were stopped (data notshown). This observation in controlled environment root boxes isconsistent with the increased root depth observed with moisture sensorsin the field and shows that deeper roots may occur at earlierdevelopmental stages, although differences in root depth were notdetected in the field experiment until after V11/V12 stage.

Although the root traits measured in the controlled environmentexperiments described in Example 14 below generally did not show asignificant difference in root depth (or only a minimal difference), thevermiculite experiment in Example 14 was performed at V3 stage beforethe difference in root depth was observed in the root box experiment inthis Example 11 (i.e., starting around V4 stage), and although theaeroponic apparatus experiment in Example 14 was performed at V5 stage,the aeroponic system does not have any plant-soil interaction (unlikethe vermiculite experiments) that might affect normal (or more natural)root growth and development.

Example 12. Transgenic Plants have Higher Stomatal Conductance in Normaland Drought Conditions and Maintain Higher Photosynthesis Capacity UnderDrought Stress

Stomatal conductance and photosynthesis levels in leaves under normaland drought conditions was also measured in the greenhouse. For thisexperiment, transgenic plants with the GA20 oxidase suppressionconstruct from Example 1 and wild-type control plants were subjected toa well watered (1500 ml water per day) or limited water/chronic drought(1000 ml water per day) treatment. Twenty (20) reps of the wild-typecontrol plants and ten (10) reps per event (two events total) for theGA20 oxidase suppression construct were subjected to the well wateredtreatment, and one-hundred and forty (140) reps of the wild-type controlplants and seventy (70) reps per event (two events total) for the GA20oxidase suppression construct were subjected to the limitedwater/chronic drought treatment. Border plants of appropriate height(hybrids for WT plants and inbreds for transgenic plants) were placedaround the perimeter of the experimental plants in the greenhouse tonormalize the effects of shading. Diurnal stomatal conductance andphotosynthesis measurements were taken in the morning and afternoon witha LI-COR® device at V12 stage per manufacturer's instructions. As shownin FIG. 12A, stomatal conductance was found to be consistently higherfor the transgenic plants under both well-watered and drought conditionsat both daily time points. Transgenic plants were also observed to haveless leaf rolling under the drought condition. As further shown in FIG.12B, a higher photosynthesis rate was also observed in response todrought conditions that did not significantly respond to increasedsunlight in the afternoon, unlike control plants that showed a drop inthe rate of photosynthesis in the afternoon particularly under droughtconditions.

These results (in combination with the separate field observationsabove) demonstrate that the transgenic plants with the GA20 oxidasesuppression construct not only had higher gas exchange andphotosynthesis in the leaf, but maintained a higher gas exchange andphotosynthesis in the leaf in response to water limiting/chronic droughtconditions. It was further observed that transgenic plants had a lowerleaf temperature than control plants (data not shown). Thus, it ispredicted that transgenic plants expressing a GA20 oxidase suppressionconstruct may have greater drought tolerance and an ability to maintainphotosynthesis under water limiting conditions as compared to controls.Without being bound by theory, it is further proposed that the deeperroots observed for transgenic plants with the GA20 oxidase suppressionconstruct (particularly during late vegetative and early reproductivestages) may contribute to the drought tolerance of these transgenicplant.

Example 13. Transgenic Plants Exhibited Reproductive Traits Comparableto Those of the Control in Greenhouse Conditions

Transgenic corn plants having the GA20 oxidase suppression constructdescribed in Example 1 and control plants were grown in pots in thegreenhouse to reproductive R1 stage, and reproductive traits weremeasured in V8 and R1 stages. Data were taken for transgenic plants oftwo transformation events (Table 10). The data are provided either interms of a difference in the number of days, or as a percent difference,for the transgenic plants as compared to a wild-type control, andsignificant changes are in bold. Trait names are defined in Examples 9and 10 above. Specific observations of the traits and trait classes offlowering, immature ear, mature ear and tassel are summarized in thetable. Overall, reproductive development in transgenic plants was nearlyequivalent to control plants with only a few slight or minor changes.

TABLE 10 Greenhouse reproductive traits of transgenic plants vs control.Class Trait Event-1 Event-2 Observations Development Plant Height

Shorter plant (R1) Leaf Tip

 1.10% Slight increase in leaf Number numbers (0.3) Flowering Days to50%

Slightly delayed pollen (R1) Silking and 50% shedding time with normalPollen Shed silking time; lower ASI Days to 50%

Pollen Shedding Days to 50%  0.40% −0.10% Visible Silk Immature ImmatureEar

Slower initiation of Ear Diameter at ear development (V8) base ImmatureEar −6.10% −4.20% Internode Length Immature Ear

Length Immature

Kernels/Row Longitudinally Mature Ear Kernels/Row   −1% −0.40% Properlydeveloped (R1) Longitudinally mature ear Kernel Row  2.20%     0% NumberTotal floret  1.10% −0.50% number Shank −3.60%  0.10% internode numberTassel Number of −5.40% −3.80% Properly developed (R1) Tassel Branchestassel but with shorter Primary Lateral

−9.10% first internode Tassel Branch Number Secondary −17.60%  −13.70% Lateral Tassel Branch Number Rachilla Floret −8.50% −0.40% Density FirstTassel

Internode Length

Example 14. Root Traits of Transgenic and Control Plants in GreenhouseConditions

Transgenic plants having the GA20 oxidase suppression constructdescribed in Example 1 and control plants were grown in the greenhousein vermiculite medium to V3 stage or in an aeroponic apparatus to V5stage. Plants were extracted and roots washed for direct or opticalimaging measurements of the root traits. Transgenic plants of 4transformation events were tested in comparison to a control.Measurement results are summarized in Table 11 and 12 for plants fromvermiculite medium growth, or in the aeroponic growth apparatus,respectively. Root Branch Point Number measures the number of rootbranch tip points of a plant through imaging of the plant root. The rootsystem image was skeletonized for the root length measurement. Up to 40images were taken at various angles around the root vertical axis andthe measurement was averaged over the images. Root Total Length measuresthe cumulative length of roots of a plant, as if the roots were alllined up in a row, through imaging of the root system of the plant. Theroot system image was skeletonized for the root length measurement. Upto 40 images were taken at various angles around the root vertical axisand measurement was averaged over the images. Data in Tables 11 and 12are the percent delta difference of the transgenic plants in comparisonto the control with significant changes presented in bold.

TABLE 11 Greenhouse root traits of transgenic plants vs control at V3,in vermiculite medium Event-1 Event-2 Event-3 Event-4 Average RootDiameter

−5.9 Root Branch Point Number

5.8

−0.4 Root Dry Weight 1 −5.6 −7.1 −5 Root Surface Area 2.2 −6.2 −6 0 RootTotal Length

−1.9 3 1.4 Plant Height

Shoot Dry Weight −3.6 −2 −4.5 −7.7 Shoot to Root Ratio −1.5 3.4 1.8 −3.4

TABLE 12 Greenhouse root traits of transgenic plants vs control at V5,in aeroponic apparatus. Event-1 Event-2 Event-3 Event-4 Root BranchPoint Number −6.18 5.01 5.63 6.38 Root Total Length −1.47 5.43 2.46 6.92Average Root Width −1.12 −5.05 −5.23 −3.56 Root Volume −1.1 −4.21 −8.47−1.09 Root Dry Weight 5.21 −7.51 −2.61 4.52 Root Surface Area −1.51 0.93−2.71 3.06 Plant Height

Shoot Dry Weight

Total Dry Weight −4.41

−8.54 −3.24 Shoot/Root Ratio

As shown in Tables 11 and 12, the transgenic plants exhibitedsignificant decrease in plant heights at V3 and V5 stages, but onlyminor variations in the overall root architecture were observed in theseexperiments between transgenic and control plants.

Example 15. Phenotypic Observations of Transgenic Plants with AlternatePromoters

In Examples 1 through 14, transgenic plants contained a GA20 oxidasesuppression element operably linked to an RTBV promoter. Corn plantswere also transformed with the same suppression element operably linkedto various other promoters, to test how different patterns of expressionof the GA20 oxidase suppression element might affect plant height andother phenotypes.

Transgenic plants (R0 plants) regenerated from explants transformed withconstructs operably linked to various promoters were observed at R5growth stage in the greenhouse, and the ears were observed after beingpeeled back for dry down. The various promoters tested are identified inTable 13. Observations were made for plants of multiple transformationevents for each construct containing a different promoter in comparisonto control plants of the same breeding line without the GA20 oxidasesuppression construct. The results of these observations are summarizedin Table 13 across transformation events for each construct.

TABLE 13 Summary of R0 observations of transgenic plants with a miRNAsuppression construct for GA20 oxidase under the control of differentpromoters. R0 plants Promoter Name Expression pattern observations RTBVpromoter vascular enhanced short; no off type CAMV e35S promoterconstitutive some short (variable); no off type Coix lacryma-jobiconstitutive some short (variable); polyubiquitin promoter no off typerice actin promoter constitutive some short (variable); no off type riceGos2 promoter constitutive some short (variable); no off type Enhancer +RTBV constitutive short; no off type promoter C1 constitutive Short cornPPDK promoter leaf enhanced, high mid-short; no off type corn FDApromoter leaf enhanced, medium some short (variable); no off type riceNadh-Gogat leaf enhanced, low mid-short; no off type promoter rice Cyp2promoter vascular enhanced some short (variable); no off type V1vascular enhanced short; no off type V2 vascular enhanced normal height;no off type V3 vascular enhanced normal height; no off-type MMV.FLTpromoter stem enhanced, high normal height; no off-type S1 stemenhanced, medium normal height; no off-type S2 stem enhanced, mediumnormal height; no off-type S3 stem enhanced, medium normal height; nooff-type SETit.lfr promoter root enhanced, high mid-short; vascularenhanced no off-type Rice Rcc3 promoter root enhanced, low normalheight; no off-type Rice Expb promoter ear enhanced, high normal height;no off-type Maize H2a promoter ear enhanced, low normal height; nooff-type

As shown in Table 13, in comparison with controls, R0 transgenic plantswith the GA20 oxidase suppression construct did not exhibit anysignificant off-types by observation for all of the promoters tested.Even expression directly in reproductive ear tissues did not cause anyobservable off-types. Plant heights were clearly decreased not only forthe RTBV promoter construct (in the previous Examples), but also fortransgenic plants having the same GA20 oxidase suppression constructoperably linked to various constitutive promoters, leaf promoters atdifferent expression levels, some vascular promoters, and a rootpromoter with a high expression level. An engineered promoter withconstitutive expression (C1) linked to the GA20 oxidase suppressionconstruct was tested and also found to cause a short stature phenotype.Similarly, at least one engineered promoter with vascular expression(V1) linked to the GA20 oxidase suppression construct was found to causea short stature phenotype, in addition to the vascular rice Cyp2promoter, although plants with two other engineered vascular promoters(V2, V3), and three engineered stem promoters (S1, S2, S3), did not havea reduced plant height. However, changing the transcriptional terminatorsequence for the GA20 oxidase suppression construct under the control ofthe RTBV promoter did not alter the short stature phenotype (not shownin Table 11). As used herein, the term “mid-short” refers to a moderatereduction in plant height (relative to the reduction in plant heightobserved with the RTBV promoter), and an observation of “some short”means that there was some variation in the amount of reduction in plantheight.

These results show that expression of the GA20 oxidase suppressionelement with constitutive promoters consistently produced a shortstature phenotype, although there was some variability in the plantheight phenotypes observed with these constitutive promoters. Likewise,a combination of the RTBV promoter with an enhancer element to convertthe pattern of expression from vascular to constitutive still produced ashort stature phenotype (indicating the sufficiency of the RTBVpromoter). A few of the vascular promoters including the RTBV promoterproduced a short stature phenotype, but a couple other engineeredvascular promoters did not produce the short stature phenotype, whichmay be attributed to a lower expression level with these promoters. Noneof the stem promoters produced a short stature phenotype, indicatingthat expression of the GA20 suppression construct in the stem was notsufficient to produce this phenotype. Surprisingly, expression of theGA20 suppression construct in the leaf consistently produced shortstature phenotypes with different levels of expression, although theresults were somewhat variable. This data indicates that the productionof active GAs in leaf tissue contributes to plant growth and ultimatelyplant height, even though such vertical growth occurs in the stem orstalk of the plant. Expression of the GA20 oxidase suppression constructwith various root promoters generally did not produce a short staturephenotype, although one root promoter did produce a moderate phenotype,which may be due to additional expression in above-ground plant tissues.

R0 plants were then self-crossed and the resulting seeds were grown inthe nursery to generate homozygous inbred progeny plants (R1 plants).Observations of R1 progeny transgenic plants with some of the promoterconstructs (at least 4 transformation events per construct;) were madeat the R1 developmental stage, in comparison to control plants of thesame breeding line without the GA20 oxidase suppression construct. Likethe R0 plants, R1 progeny plants expressing the GA20 oxidase suppressionconstruct with each of the RTBV, CAMV e35S, and Coix lacryma-jobipolyubiquitin promoters were also found to have a short stature,semi-dwarf phenotype without any significant off-types observed.

Example 16. Phenotypic Observations of Transgenic Corn Plants withConstructs Targeting Different GA Oxidase Genes

The Examples above demonstrate that a miRNA-expressing constructtargeting the GA20 oxidase_3 and GA20 oxidase_5 genes for suppression,and operably linked to a plant-expressible vascular, constitutive and/orleaf promoter, may be used to generate a short stature, semi-dwarf cornplant. To test how targeting different GA20 or GA3 oxidase genes, ordifferent portions of the GA20 oxidase_3 and/or GA20 oxidase_5 genes,for suppression might affect plant height, several constructs weregenerated and transformed into corn plants. Constructs were also madewith the same targeting sequence as in the above Examples, but with adifferent miRNA backbone sequence (two from corn miRNAs, one from asoybean miRNA, and one from a cotton miRNA—the construct in the aboveExamples used a rice miRNA backbone sequence). Table 14 provides a listof these additional suppression constructs, along with observations oftransgenic R0 plants comprising these constructs in the greenhouse (incomparison to wild-type control plants). Constructs targeting (i) GA20oxidase_1/GA20 oxidase_2, (ii) GA20 oxidase_3/GA20 oxidase_9, (iii) GA20oxidase_7/GA20 oxidase_8, and (iv) GA20 oxidase_3/GA20 oxidase_5 (withdifferent miRNA backbones), each encoded a miRNA with a single targetingsequence complementary to both gene targets, whereas the stacks of (i)the individual GA20 oxidase_3 and GA20 oxidase_5 targeting sequences,(ii) the individual GA20 oxidase_4 and GA20 oxidase_6 targetingsequences, and (iii) the individual GA20 oxidase_4 and GA20 oxidase_7/8targeting sequences, were each expressed as a single pre-miRNA with thetwo targeting sequences arranged in tandem that become cleaved andseparated into two mature miRNAs. Table 14 provides the miRNA targetingsequence and the cDNA sequence complementary to the miRNA targetingsequence. For the GA20 oxidase_1/GA20 oxidase_2 construct, the asterisk(*) indicates that the alignment length between the targeting sequenceof the miRNA and the mRNA target or recognition site was shorter (17 vs.20 nucleotides) for GA20 oxidase_1 than for GA20 oxidase_2. Similarlyfor the GA20 oxidase_3/GA20 oxidase_9 construct, the asterisk (*)indicates that the alignment length between the targeting sequence ofthe miRNA and the mRNA target or recognition site was shorter (17 vs. 20nucleotides) for GA20 oxidase_9 than for GA20 oxidase_3. For each of theconstructs listed in Table 14, no significant off-types were observed,apart from the observations provided in the table.

TABLE 14 Summary of R0 observations of transgenic plants with miRNAsuppression constructs targeting different GA oxidase genes. cDNA miRNAmRNA Target Targeting Targeted Gene(s) Targeted Sequence Sequence(Construct/Promoter) Area (SEQ ID NO) (SEQ ID NO) Observations GA20oxidase_1 and 1: exon* 47 48 All events tall (WT) GA20 oxidase_2 2: exon(RTBV promoter) GA20 oxidase_3 and 3: exon 49 50 All events tall (WT)GA20 oxidase_9 9: exon* (RTBV promoter) GA20 oxidase_7 and exon 51 52All events - tall (WT) GA20 oxidase_8 (RTBV promoter) GA20 oxidase_3 UTR53 54 Events slightly shorter (Individual; RTBV (~6 inches vs. WT) and35S promoter) GA20 oxidase_5 UTR 55 56 All events - tall (WT)(Individual; RTBV and 35S promoter) GA20 oxidase_3 and 3: UTR 53 54 Allevents - shorter GA20 oxidase_5 5: UTR 55 56 (Individuals; Tandem stack)GA20 oxidase_3 and 3/5: exons 39 40 All events/constructs - GA20oxidase_5 shorter (Different miRNA backbones) (RTBV promoter) GA3oxidase_1 UTR 57 58 All events - tall (WT) (RTBV promoter) (only 3events observed) GA3 oxidase_1 UTR 57 58 Some events - shorter (CAMVe35S promoter) GA3 oxidase_2 exon 59 60 All events - shorter (RTBVpromoter) (darker green leaves) GA3 oxidase_2 exon 59 60 Some eventsshorter (CAMV e35S promoter) GA20 oxidase_4 and 4: exon 61 62 Someevents - GA20 oxidase_6 6: exon 63 64 moderately shorter (~20%)(Individuals; Tandem stack) GA20 oxidase_4 and 4: exon 61 62 Someevents - GA20 oxidase_7/8 7/8: exon 51 52 moderately shorter (~20%)(Individuals; Tandem stack)

The observations summarized in Table 14 demonstrate that targeting ofseveral other GA20 oxidase genes did not produce a short stature,semi-dwarf phenotype. None of the constructs targeting (i) the relatedGA20 oxidase_1 and GA20 oxidase_2 genes, (ii) the related GA20 oxidase_3and GA20 oxidase_9 genes, (iii) the related GA20 oxidase_7 and GA20oxidase_8 genes, or (iv) the GA20 oxidase_9 gene alone produced anobservable short stature, semi-dwarf phenotype in R0 plants. Incontrast, those constructs encoding a single miRNA jointly targeting theGA20 oxidase_3 and GA20 oxidase_5 genes in transgenic R0 and R1 plantsdid produce a short stature, semi-dwarf phenotype, even if a differenttranscriptional termination sequence or different miRNA backbones areused (total of 5 miRNA backbone sequences tested). In addition,targeting different sequences of the GA20 oxidase_3 and GA20 oxidase_5genes still produced semi-dwarf plants. Interestingly, suppressionconstructs that were designed to target either of the GA20 oxidase_3 andGA20 oxidase_5 genes individually did not produce a short stature,semi-dwarf phenotype, unlike constructs jointly targeting the GA20oxidase_3 and GA20 oxidase_5 genes, although the construct individuallytargeting the GA20 oxidase_3 gene did produce a slight reduction inplant height. However, transgenic plants having a tandem vector stack ofthe suppression constructs individually targeting the GA20 oxidase_3 andGA20 oxidase_5 genes did produce a short stature, semi-dwarf phenotypesimilar to constructs encoding a single miRNA jointly targeting the GA20oxidase_3 and GA20 oxidase_5 genes. These data demonstrate that a shortstature, semi-dwarf phenotype is observed with constructs targeting bothof the GA20 oxidase_3 and GA20 oxidase_5 genes, but the full semi-dwarfphenotype is not observed with targeting of the GA20 oxidase_3 and GA20oxidase_5 genes individually for suppression (only a slight reduction inheight with targeting GA20 oxidase_3, and no plant height phenotypeobserved with targeting GA20 oxidase_5). Moreover, no plant heightphenotype was observed with targeting the GA20 oxidase_1, GA20oxidase_2, GA20 oxidase_6, GA20 oxidase_7, GA20 oxidase_8, and/or GA20oxidase_9 gene(s) as described.

Apart from the GA20 oxidase_3 and GA20 oxidase_5 genes, a moderatereduction in plant height was observed in R0 transgenic plants with asuppression construct comprising two targeting sequences in tandemcomplementary to jointly target (i) the GA20 oxidase_4 and GA20oxidase_6 genes, or (ii) the GA20 oxidase_4, GA20 oxidase_7 and GA20oxidase_8 genes—one of the two targeting sequences targets both the GA20oxidase_7 and GA20 oxidase_8 genes. Given that a separate construct thattargets the GA20 oxidase_7 and GA20 oxidase_8 genes did not produce aplant height phenotype, and the suppression construct targeting the GA20oxidase_4 and GA20 oxidase_6 genes produced a plant height phenotypethat was similar to the suppression construct targeting the GA20oxidase_4, GA20 oxidase_7 and GA20 oxidase_8 genes, it is believed thattargeting of the GA20 oxidase_4 gene is largely (if not fully)responsible for the plant height phenotype observed in these transgenicplants. Furthermore, transgenic corn plants with constructs targetingthe GA3 oxidase_1 or GA3 oxidase_2 genes also displayed a reduction inplant height, although there was some variability in this phenotypedepending on the constitutive promoter. Thus, in addition the GA20oxidase_3 and GA20 oxidase_5 genes, the GA20 oxidase_4, GA3 oxidase_1,and GA3 oxidase_2 genes may also be targeted for suppression to produceshort stature, semi-dwarf plants.

Example 17. Phenotypic Observations of Corn Plants Having an Edited GA20Oxidase_3 or GA20 Oxidase_5 Gene

In addition to the above suppression constructs, several genome-editedmutations were created in the endogenous GA20 oxidase_3 and GA20oxidase_5 genes in corn plants to test for the phenotypic effect ofknocking out each of these genes. A series of ten single-chain guide RNA(sgRNAs) encoding targeting constructs were created for each of the GA20oxidase_3 and GA20 oxidase_5 genes that target different positions alongthe genomic sequence for each gene. An additional series of ten sgRNAswere created that each target both of the GA20 oxidase_3 and GA20oxidase_5 genes, at similar or different positions along the genomicsequence for each gene. Targeted genome edits were made by deliveringthe sgRNA along with expression of a Cas9 protein to corn explants tocause a DSB or nick to occur at or near the genomic target site for thegRNA, which may then be imperfectly repaired to introduce a mutation ator near the target site. The presence of a mutation was subsequentlyconfirmed by RFLP analysis and/or sequencing of plants. Table 15 belowprovides a list of the guide RNA (gRNA) constructs that were tested,which may be used for genome editing of one or both of the GA20oxidase_3 and GA20 oxidase_5 gene(s) with a RNA-guided endonuclease.These guide RNA constructs are generally designed to target the codingsequences of the GA20 oxidase_3 and GA20 oxidase_5 genes, but some ofthe joint targeting constructs may instead target a UTR sequence of oneof the two genes. These gRNAs may be used with a suitable endonucleaseto produce a double stranded break (DSB) or nick in the genome at ornear the genomic target site of the respective gRNA, which may beimperfectly repaired to produce a mutation (e.g., an insertion,deletion, substitution, etc.). Transgenic plants that were homozygousfor an edited GA20 oxidase_3 gene or homozygous for an edited GA20oxidase_5 gene were generated from a few of the constructs (see boldtext). Events were also generated from constructs targeting both genesfor editing. For the constructs jointly targeting the GA20 oxidase_3 andGA20 oxidase_5 genes, the coding sequence (CDS) coordinates are providedin reference to one of the two genes as indicated in parenthesis. Table15 further shows which constructs produced gene editing events, whetherthose events were homozygous or heterozygous in the R0 plants, and the±numbers in parenthesis indicate the likely sequence change with themutation (e.g., +1 means an insertion of 1 nucleotide, etc., and largeror more complicated Indels are labeled “del.” or insert.”). For stackedtargeting of GA20 oxidase_3 and GA20 oxidase_5, the identity of themutated gene is also provided in parenthesis. Consistent with theresults for the suppression constructs, transgenic plants homozygous foran edited GA20 oxidase_3 or GA20 oxidase_5 gene did not have a shortstature, semi-dwarf phenotype and had a normal plant height relative tocontrol plants (See constructs GA20 oxidase_3-D and GA20 oxidase_3-G,and constructs GA20 oxidase_5-B and GA20 oxidase_5-G in Table 15),indicating that knockout of only one of these genes is not sufficient toproduce the semi-dwarf phenotype.

TABLE 15 Guide RNAs (gRNAs) targeting GA20 oxidase_3 and GA oxidase_5genes for editing. gRNA Targeting Sequence Gene CDS gRNA Gene Target(SEQ ID NO) coordinates Events Generated GA20 oxidase_3-A 138 552-572 —GA20 oxidase_3-B 139 879-899 — GA20 oxidase_3-C 140 147-167 — GA20oxidase_3-D 141 526-546 1. homozygous (−1) 2. heterozygous (−1) 3.bi-allelic (−2, +1) GA20 oxidase_3-E 142 446-466 — GA20 oxidase_3-F 1432227-2247 — GA20 oxidase_3-G 144 548-568 1. homozygous (+1) 2.heterozygous (−1) 3. bi-allelic (+1, −1) GA20 oxidase_3-H 145 547-567 —GA20 oxidase_3-I 146 43-63 — GA20 oxidase_3-J 147 548-567 — GA20oxidase_5-A 148   356-376 (+) 1. heterozygous (−1) GA20 oxidase_5-B 149 99-119 1. homozygous (−1) 2. heterozygous (+1) 3. heterozygous (+1, −7)4. heterozygous (−3, −1) GA20 oxidase_5-C 150 369-389 — GA20 oxidase_5-D151 48-68 — GA20 oxidase_5-E 152   356-376 (−) — GA20 oxidase_5-F 153748-768 1. heterozygous (−1, +1) GA20 oxidase_5-G 154 770-790 1.homozygous (−1) 2. homozygous (−1) GA20 oxidase_5-H 155 10-30 — GA20oxidase_5-I 156 262-282 — GA20 oxidase_5-J 157 768-788 — GA20oxidase_3/5-A 158 290 . . . 310 — (GA20 Ox_3) GA20 oxidase_3/5-B 159 289. . . 309 — (GA20 Ox_3) GA20 oxidase_3/5-C 160 270 . . . 290 — (GA20Ox_5) GA20 oxidase_3/5-D 161 49 . . . 69 — (GA20 Ox_3) GA20oxidase_3/5-E 162 265 . . . 285 1. heterozygous (GA20 Ox_5) (Ox5, +1)GA20 oxidase_3/5-F 163 419 . . . 439 1. hetero (Ox3, +1, −1) (GA20 Ox_3)hetero (Ox5, +1, del.) 2. hetero (Ox3, +1, del.) hetero (Ox5, +1,insert.) GA20 oxidase_3/5-G 164 110 . . . 130 — (GA20 Ox_3) GA20oxidase_3/5-H 165 634 . . . 654 — (GA20 Ox_5) GA20 oxidase_3/5-I 166  98. . . 118 — (GA20 Ox_5) GA20 oxidase_3/5-J 167 517 . . . 537 — (GA20Ox_5)

Example 18. Suppression Construct Targeting GA20 Oxidase_3 and GA20Oxidase_5 Genes Reduces GA20 Oxidase Transcript and Active GA Levels inthe Plant

To determine how GA20 oxidase transcript levels were affected intransgenic plants with the suppression construct targeting the GA20oxidase_3 and GA20 oxidase_5 genes, whole tissues from various parts oftransgenic plants grown in the greenhouse were taken at differentvegetative stages (V3, V8, and V14), and mRNA transcript levels for eachof the GA20 oxidase genes were analyzed using a TaqMan® assay. For theseexperiments, total RNA was extracted using a Direct-Zol RNA extractionkit from Zymo Research™ and treated with Turbo™ DNase to reduce genomicDNA contamination. RNA was then reverse transcribed to generatedouble-stranded cDNA. Reverse transcription quantitative PCR wasperformed with gene specific primers and FAM labeled TaqMan® probes onthe Bio-Rad® CFX96 Real Time System. Quality control metrics werecalculated using tissue specific standards to determine qPCR efficiencyand total RNA that had not undergone reverse transcription to accountfor residual genomic DNA contamination. The difference between cyclethreshold values for genes of interest versus normalizer genesdetermined the relative quantity of each gene transcript in each tissue.This relative quantity was calculated using either one (18S) or thegeometric mean of two (18S and ELF1A) normalizer genes.

In this experiment, the level of the GA20 oxidase_3 transcript wasreduced in most of the vegetative tissues at these stages, includingleaf and stem tissue at V3, internode tissue at V8, and leaf andinternode tissue at V14, although the level of GA20 oxidase_3 transcriptin V3 root and V8 leaf appeared unchanged (data not shown). Furthermore,the level of GA20 oxidase_5 transcript for this experiment was generallyunchanged in the vegetative tissues tested (data not shown), althoughthe level of expression of the GA20 oxidase_5 transcript was relativelylow in these tissues. Neither GA20 oxidase_3 nor GA20 oxidase_5 weresignificantly reduced in root tissue samples of transgenic plants. Eachof the other GA20 oxidase genes (i.e., the 1, 2, 4 and 6-9 subtypes)were generally unchanged or increased in some tissues of the transgenicplants.

A similar experiment was conducted with reproductive tissues fromtransgenic plants expressing the same suppression construct. Wholetissues from various parts of transgenic plants grown in the greenhousewere taken at different reproductive stages (R1 and R3), and mRNAtranscript levels for each of the GA20 oxidase genes were analyzed usinga TaqMan® assay. In this experiment, the levels of GA20 oxidase_3 andGA20 oxidase_5 transcripts were mostly unchanged in R1 leaf, ear, tasseland internode and R3 leaf and internode, relative to controls (data notshown). Results for the other GA20 oxidase genes were mostly mixed orneutral (data not shown).

These data show that the level of GA20 oxidase_3 transcripts intransgenic corn plants during vegetative stages was generally reduced inthis experiment, but appears mostly unchanged relative to control planttissues during later reproductive stages. Although a clear reduction inthe level of GA20 oxidase_5 gene transcripts was not generally observedin these transgenic plant tissues, the expression level of this gene wasrelatively low. Thus, changes in its expression level may have beendifficult to detect with this method. In addition, the suppressionconstruct appears to be specific to the targeted GA20 oxidase genessince no consistent reduction in expression level was observed in thisexperiment for any of the other GA20 oxidase genes.

In a separate experiment, GA20 oxidase expression levels were determinedin stem tissues of transgenic plants expressing the suppressionconstruct from the prior Examples (targeting the GA20 oxidase_3 and GA20oxidase_5 genes for suppression under the control of the RTBV promoter),in comparison to a wild-type control. Tissue samples were taken fromV3-V6 stems/stalks and parts of those stems were further dissected toseparate vascular and non-vascular tissues to determine differentialexpression levels among these tissues. Transcript expression levels weredetermined using a RNA sequencing (RNA-Seq) approach for quantitativecomparison between transgenic and wild-type plant tissues. The datapresented in FIG. 13A are generated from transgenic plants having one oftwo events and wild type control plants having one of two germplasms,with each bar in FIG. 13A representing one of the two transgenic eventsor germplasms, respectively. For these experiments, individual vascularbundles were separated from the remaining stem/stalk tissue of thesamples and subjected to separate analysis. As shown in FIG. 13A, themiRNA expressed by the suppression construct was detected in bulk plantstem tissue (“bulk”; i.e., without separation of vascular andnon-vascular tissues), as well as in separated vascular (“Vasc”) andnon-vascular (“Non-Vasc”) tissues from the bulk stem/stalk sample.However, the expression level of the miRNA was much higher in vasculartissue than in non-vascular tissue indicating the vascular expressionpattern of the RTBV promoter.

The bulk stem/stalk samples and the separated vascular and non-vascularsamples were also analyzed in a similar RNA-Seq experiment to measureand compare the levels of GA20 oxidase_3 and GA20 oxidase_5 genetranscripts in transgenic versus wild-type control plants (along withother GA20 oxidase genes), although only one wild-type sample is shownfor each tissue type. For these experiments, stalk tissue from controlor transgenic plants (two events) were sectioned to separate vascularbundles and non-vascular tissues as described above. Total sRNA and mRNAwere sequenced for each sample, and data was analyzed and compared usingprinciple component analysis.

As shown in FIG. 13B, transcript levels of the GA20 oxidase_3 gene weresignificantly reduced in bulk stem tissue (Bulk) and separated stemvascular tissues (Vasc) of transgenic plants (TG) relative to wild-typecontrols (WT), but appeared unchanged in separated non-vascular(Non-Vasc) tissue. However, transcript levels of the GA20 oxidase_5 genewere significantly reduced in bulk stem tissue (Bulk), but relativelyunchanged in separated vascular (Vasc) and non-vascular (Non-Vasc)tissues of transgenic plants, although there was a downward trend linefor the GA20 oxidase_5 transcript in vascular (Vasc) tissue samples fromtransgenic plants. The level of expression of the GA20 oxidase_5 genewas low, particularly in non-vascular tissues. All other GA20 oxidasegenes did not show a significant reduction in their transcript levels inthe transgenic plant tissues analyzed, although a couple GA20 oxidasegenes did show a slight upward trend in their level of expression. Thisdata further demonstrates that the expression levels of the GA20oxidase_3 and GA20 oxidase_5 genes are decreased to varying extents inone or more tissues of transgenic plants having the suppressionconstruct relative to controls. Indeed, the higher expression of themiRNA and greater suppression of the endogenous GA20 oxidase_3 gene invascular tissues is consistent with the vascular pattern of expressionof the RTBV promoter, and perhaps the higher levels of GA20 oxidase_3gene expression in vascular versus non-vascular tissues of wild-typeplants. A similar pattern is also observed for the GA20 oxidase_5 gene,although not as pronounced as the GA20 oxidase_3 gene between vascularand non-vascular tissues.

The short stature, semi-dwarf phenotype observed with GA20 oxidasesuppression in transgenic plants is likely mediated by a reduction inthe level of active GAs present in the stem or internode tissues and/orin plant tissues that produce active GAs. To determine the levels ofactive GAs (particularly G1, G3 and G4) relative to other inactive formsof the hormone, GA levels were measured in different tissue samplestaken from transgenic and wild-type control plants at different stagesof development. For these experiments, fresh frozen samples for eachtissue were milled and dispensed into 96 well glass tubes along withinternal standards. Samples were extracted using methanol:water:aceticacid (80:19:1 v/v/v) solvent two times for 4 hours at 4° C. Solvent wasevaporated from the extract to near dryness using multi-channel SPE withnitrogen. Samples were further purified using a SPE cartridge. Afterpurification, samples were run using standard LC-MS/MS method withShimadzu® Nexera® UPLC and SCIEX® triple quad 5500 mass specinstrumentation. Chromatographs were analyzed and quantified usinginternal standards.

Two sets of experiments were performed with samples taken from varioustissues of vegetative stage plants. As shown in Table 16 for oneexperiment in the greenhouse, reduced levels of active GAs (GA1, GA3,and GA4) were observed in various tissues of transgenic plants atdifferent vegetative stages. The data in Table 16 is displayed as thenumber of transgenic plants having a significant change in the amount ofeach GA hormone for a given tissue (“U”=up or increased; “D”=down ordecreased; “N”=neutral or no change; and “T”=total number of plants).The GAs that showed at least a partial reduction in tissue samples arepresented in bold. Active GA1 was reduced in leaf and internode tissuesat V8 stage and internode tissue at V14 stage, and active GA4 wasreduced in V3 stem and V8 and V14 internode. However, active GA3 was notobservably reduced in this experiment. Other inactive forms of GAs werealtered in various tissues of transgenic plants as shown in Table 16. Ingeneral, GAs that are downstream of GA20 oxidase genes in thegibberellic acid pathway (e.g., GA9, GA20, and GA34) tended to bereduced, whereas GAs that are upstream of GA20 oxidase genes tended tobe higher (e.g., GA12 and GA53), which may be due to the lower activityof GA oxidase genes causing the precursor GAs upstream to accumulate.This data is consistent with suppression of GA20 oxidase activity inthese tissues and lower levels of active GA hormones in the stem andleaf of transgenic plants.

In a separate experiment, similar measurements of GA hormones were takenfrom various plant tissues during vegetative stages of development. Asshown in in Table 17 for an experiment using tissues taken from plantsin the greenhouse and field, reduced levels of one or more active GAs(GA1, GA3, and GA4) were observed in the leaf and internode oftransgenic plants at V3 and V8 stages. The leaf samples at V8 stage forthis experiment were taken from plants in the field, unlike the othersamples taken from plants in the greenhouse. The data in Table 17 isdisplayed in a similar manner as described for Table 16. Other inactiveforms of GAs were altered in various tissues of transgenic plants asshown in Table 17. Similar to the observations above, GAs that aredownstream of GA20 oxidase genes in the gibberellic acid pathway (e.g.,GA9, GA20, and GA34) tended to be reduced, whereas GAs that are upstreamof GA20 oxidase genes tended to be higher (e.g., GA12 and GA53). Thisdata is again consistent with suppression of GA20 oxidase activity inthese tissues and lower levels of active GA hormones in the stem andleaf of transgenic plants.

TABLE 16 Change in GA hormone levels in tissues of transgenic cornplants expressing a GA20 oxidase suppression construct in thegreenhouse. Stage: V3 V8 V14 Tissue: Leaf Stem Root Leaf Internode LeafInternode Tassel GA1 2N/2T 2N/2T 2N/2T 2D/2T 1D/1N/2T 2N/2T 2D/2T 2N/2TGA3 2N/2T 2N/2T 2N/2T 2N/2T 2N/2T 2N/2T 2N/2T 2N/2T GA4 2N/2T 2D/2T2N/2T 2N/2T 2D/2T 2U/2T 2D/2T 2N/2T GA8 1U/1N/2T 2N/2T 1U/1N/2T 2N/2T2D/2T 1D/1N/2T 1D/1N/2T 1U/1N/2T GA9 2N/2T 2D/2T 2N/2T 2N/2T 2D/2T 2U/2T2D/2T 1D/1N/2T GA12 1D/1N/2T 2U/2T 2N/2T 2U/2T 2N/2T 1U/1N/2T 2N/2T2N/2T GA20 2D/2T 2N/2T 2N/2T 2D/2T 2D/2T 2D/2T 1D/1N/2T 2N/2T GA34 2N/2T2D/2T 2N/2T 2N/2T 2D/2T 2N/2T 2D/2T 2N/2T GA53 2U/2T 2U/2T 2N/2T 2U/2T2N/2T 2U/2T 1U/1N/2T 1U/1N/2T

TABLE 17 Change in GA hormone levels in tissues of transgenic cornplants expressing a GA20 oxidase suppression construct in the greenhouse(GH) or field. Stage: V3 V8 Tissue: Leaf (GH) Root (GH) Internode (GH)Leaf (Field) GA1 3D/1N/4T 2D/1U/1N/4T 3D/1N/4T 7D/1N/8T GA3 3D/1N/4T4N/4T 3D/1N/4T 7D/1N/8T GA4 4N/4T 4N/4T 4D/4T 8D/8T GA8 4N/4T 4N/4T4N/4T 4N/4T GA9 4D/4T 4N/4T 4D/4T 5U/3N/8T GA12 ND ND ND 7U/1N/8T GA204D/4T 1D/3N/4T 4D/4T 8D/8T GA34 1U/3N/4T 4D/4T 4D/4T 4U/4N/8T GA53 4U/4T2U/2N/4T 1D/3N/4T 8U/8T

Suppression of the GA20 oxidase_3 and GA20 oxidase_5 genes in transgeniccorn plants reduces the levels of targeted GA oxidase transcripts invarious tissues including the stem, internode, vascular tissues andleaves, and suppression of these GA20 oxidase genes is furtherassociated with reduced levels of active GAs in tissues of thetransgenic plant including the stem and internode, which is the site ofaction for affecting plant growth during vegetative stages andultimately plant height by later vegetative and reproductive stages.Similar to observations that GA20 oxidase transcript levels are mostlyunchanged or mixed in reproductive stage tissues, the levels of GAhormones including active GAs are also mostly unchanged or mixed inreproductive stage tissues (data not shown).

Having described the present disclosure in detail, it will be apparentthat modifications, variations, and equivalent embodiments are possiblewithout departing from the spirit and scope of the present disclosure asdescribed herein and in the appended claims. Furthermore, it should beappreciated that all examples in the present disclosure are provided asnon-limiting examples.

1.-110. (canceled)
 111. A recombinant DNA construct comprising atranscribable DNA sequence encoding a non-coding RNA molecule, whereinthe non-coding RNA molecule comprises a targeting sequence that is: (a)at least 80% complementary to at least 19 consecutive nucleotides of afirst mRNA molecule encoding a first endogenous gibberellin (GA) oxidaseprotein in a corn plant, the first endogenous GA oxidase protein beingat least 90% identical to SEQ ID NO: 9; and (b) at least 80%complementary to at least 19 consecutive nucleotides of a second mRNAmolecule encoding a second endogenous GA oxidase protein in a cornplant, the second endogenous GA oxidase protein being at least 90%identical to SEQ ID NO: 15; wherein the transcribable DNA sequence isoperably linked to a constitutive promoter.
 112. The recombinant DNAconstruct of claim 111, wherein the non-coding RNA molecule reduces theexpression levels of the first and second mRNA molecules in at least onetissue of a transgenic corn plant comprising the recombinant DNAconstruct, relative to a control plant, when the non-coding RNA moleculeis expressed in the transgenic corn plant.
 113. The recombinant DNAconstruct of claim 111, wherein the non-coding RNA molecule reduces theexpression levels of the first and second endogenous GA oxidase proteinsin at least one tissue of a transgenic corn plant comprising therecombinant DNA construct, relative to a control plant, when thenon-coding RNA molecule is expressed in the transgenic corn plant. 114.The recombinant DNA construct of claim 111, wherein the targetingsequence of the non-coding RNA molecule is at least 90% complementary toat least 19 consecutive nucleotides of a sequence selected from thegroup consisting of SEQ ID NOs: 7, 8, 13, and
 14. 115. The recombinantDNA construct of claim 111, wherein the targeting sequence of thenon-coding RNA molecule is at least 90% complementary to at least 19consecutive nucleotides of the first mRNA molecule encoding the firstendogenous GA20 oxidase protein.
 116. The recombinant DNA construct ofclaim 113, wherein the targeting sequence of the non-coding RNA moleculeis 100% complementary to at least 19 consecutive nucleotides of thefirst mRNA molecule encoding the first endogenous GA20 oxidase protein.117. The recombinant DNA construct of claim 113, wherein the targetingsequence of the non-coding RNA molecule is at least 90% complementary toat least 19 consecutive nucleotides of SEQ ID NO: 7 or
 8. 118. Therecombinant DNA construct of claim 111, wherein the targeting sequenceof the non-coding RNA molecule is at least 90% complementary to at least19 consecutive nucleotides of the second mRNA molecule encoding thesecond endogenous GA20 oxidase protein.
 119. The recombinant DNAconstruct of claim 116, wherein the targeting sequence of the non-codingRNA molecule is 100% complementary to at least 19 consecutivenucleotides of the second mRNA molecule encoding the second endogenousGA20 oxidase protein.
 120. The recombinant DNA construct of claim 116,wherein the targeting sequence of the non-coding RNA molecule is atleast 90% complementary to at least 19 consecutive nucleotides of SEQ IDNO: 13 or
 14. 121. The recombinant DNA construct of claim 111, whereinthe targeting sequence of the non-coding RNA molecule is at least 90%complementary to at least 21 consecutive nucleotides of the first mRNAmolecule encoding the first endogenous GA20 oxidase protein, and is atleast 90% complementary to at least 21 consecutive nucleotides of thesecond mRNA molecule encoding the second GA20 oxidase protein.
 122. Therecombinant DNA construct of claim 111, wherein the targeting sequenceof the non-coding RNA molecule is 100% complementary to at least 21consecutive nucleotides of the first mRNA molecule encoding the firstendogenous GA20 oxidase protein, and is 100% complementary to at least21 consecutive nucleotides of the second mRNA molecule encoding thesecond GA20 oxidase protein.
 123. The recombinant DNA construct of claim111, wherein the non-coding RNA molecule encoded by the transcribableDNA sequence is a precursor miRNA or siRNA that is processed or cleavedin a plant cell to form a mature miRNA or siRNA.
 124. The recombinantDNA construct of claim 111, wherein the constitutive promoter isselected from the group consisting of: an actin promoter, a CaMV 35S or19S promoter, a plant ubiquitin promoter, a plant Gos2 promoter, a FMVpromoter, a CMV promoter, a MMV promoter, a PCLSV promoter, an Emupromoter, a tubulin promoter, a nopaline synthase promoter, an octopinesynthase promoter, a mannopine synthase promoter, and a maize alcoholdehydrogenase promoter.
 125. The recombinant DNA construct of claim 111,wherein the constitutive promoter comprises a DNA sequence that is atleast 80% identical to SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82,or SEQ ID NO:
 83. 126. The recombinant DNA construct of claim 111,wherein the constitutive promoter comprises a DNA sequence that is atleast 90% identical to SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82,or SEQ ID NO:
 83. 127. The recombinant DNA construct of claim 111,wherein the constitutive promoter comprises a DNA sequence that is 100%identical to SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78,SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, or SEQ IDNO:
 83. 128. A transgenic corn plant comprising a recombinant DNAconstruct comprising a transcribable DNA sequence encoding a non-codingRNA molecule, wherein the non-coding RNA molecule comprises a targetingsequence that is: (a) at least 80% complementary to at least 19consecutive nucleotides of a first mRNA molecule encoding a firstendogenous GA oxidase protein in a corn plant, the first endogenous GAoxidase protein being at least 90% identical to SEQ ID NO: 9; and (b) atleast 80% complementary to at least 19 consecutive nucleotides of asecond mRNA molecule encoding a second endogenous GA oxidase protein ina corn plant, the second endogenous GA oxidase protein being at least90% identical to SEQ ID NO: 15; wherein the transcribable DNA sequenceis operably linked to a constitutive promoter.
 129. The transgenic cornplant of claim 128, wherein the expression levels of the first andsecond mRNA molecules are reduced in at least one tissue of thetransgenic corn plant relative to a control plant.
 130. The transgeniccorn plant of claim 128, wherein the expression levels of the first andsecond endogenous GA oxidase proteins in at least one tissue of thetransgenic corn plant relative to a control plant.
 131. The transgeniccorn plant of claim 128, wherein the transgenic corn plant has a shorterplant height relative to a control plant.
 132. The transgenic corn plantof claim 128, wherein the height of the transgenic plant is at least 10%shorter than a control plant.
 133. The transgenic corn plant of claim128, wherein the height of the transgenic plant is at least 20% shorterthan a control plant.
 134. The transgenic corn plant of claim 128,wherein the height of the transgenic plant is at least 30% shorter thana control plant.
 135. The transgenic corn plant of claim 128, whereinthe height of the transgenic plant is at least 40% shorter than acontrol plant.
 136. The transgenic corn plant of claim 128, whereinexpression of the non-coding RNA molecule in the corn plant reduces thelevel of one or more active GAs in the corn plant as compared to acontrol plant.
 137. The transgenic corn plant of claim 128, wherein thelevel of one or more active GAs in at least one internode tissue of thestem or stalk of the transgenic corn plant is lower than the sameinternode tissue of a control plant.
 138. The transgenic corn plant ofclaim 128, wherein the transgenic plant has increased lodging resistancerelative to a control plant.
 139. The transgenic corn plant of claim128, wherein the transgenic plant has reduced green snap relative to acontrol plant.
 140. The transgenic corn plant of claim 128, wherein thetransgenic plant has increased harvest index relative to a controlplant.
 141. The transgenic corn plant of claim 128, wherein thetransgenic plant has deeper roots relative to a control plant.
 142. Thetransgenic corn plant of claim 128, wherein the transgenic plant has oneor more of the following traits relative to a control plant: increasedleaf area, earlier canopy closure, higher stomatal conductance, lowerear height, increased foliar water content, improved drought tolerance,and reduced anthocyanin content or area in leaves.
 143. The transgeniccorn plant of claim 128, wherein the transgenic plant has one or more ofthe following traits relative to a control plant: increased ear weight,increased yield, increased seed number, and increased seed weight. 144.The transgenic corn plant of claim 128, wherein the stalk or stemdiameter of the transgenic plant at one or more stem internodes isgreater than the stalk or stem diameter at the same one or moreinternodes of a control plant.
 145. The transgenic corn plant of claim128, wherein the transgenic plant does not have any significantoff-types in at least one female organ or ear.
 146. The transgenic cornplant of claim 128, wherein the stalk or stem diameter of the transgeniccorn plant at one or more of the first, second, third, and/or fourthinternode below the ear is at least 5% greater than the same internodeof a control plant.
 147. The transgenic corn plant of claim 128, whereinthe level of one or more active GAs in at least one internode tissue ofthe stem or stalk of the transgenic plant is lower than the sameinternode tissue of a control plant.
 148. The transgenic corn plant ofclaim 128, wherein the targeting sequence of the non-coding RNA moleculeis at least 90% complementary to at least 19 consecutive nucleotides ofthe first mRNA molecule encoding the first endogenous GA20 oxidaseprotein.
 149. The transgenic corn plant of claim 128, wherein thetargeting sequence of the non-coding RNA molecule is 100% complementaryto at least 19 consecutive nucleotides of the first mRNA moleculeencoding the first endogenous GA20 oxidase protein.
 150. The transgeniccorn plant of claim 128, wherein the targeting sequence of thenon-coding RNA molecule is at least 90% complementary to at least 19consecutive nucleotides of SEQ ID NO: 7 or
 8. 151. The transgenic cornplant of claim 128, wherein the targeting sequence of the non-coding RNAmolecule is at least 90% complementary to at least 19 consecutivenucleotides of the second mRNA molecule encoding the second endogenousGA20 oxidase protein.
 152. The transgenic corn plant of claim 128,wherein the targeting sequence of the non-coding RNA molecule is 100%complementary to at least 19 consecutive nucleotides of the second mRNAmolecule encoding the second endogenous GA20 oxidase protein.
 153. Thetransgenic corn plant of claim 128, wherein the targeting sequence ofthe non-coding RNA molecule is at least 90% complementary to at least 19consecutive nucleotides of SEQ ID NO: 13 or
 14. 154. The transgenic cornplant of claim 128, wherein the targeting sequence of the non-coding RNAmolecule is at least 90% complementary to at least 21 consecutivenucleotides of the first mRNA molecule encoding the first endogenousGA20 oxidase protein and is at least 90% complementary to at least 21consecutive nucleotides of the second mRNA molecule encoding the secondendogenous GA20 oxidase protein.
 155. The transgenic corn plant of claim128, wherein the targeting sequence of the non-coding RNA molecule is100% complementary to at least 21 consecutive nucleotides of the firstmRNA molecule encoding the first endogenous GA20 oxidase protein and is100% complementary to at least 21 consecutive nucleotides of the secondmRNA molecule encoding the second GA20 oxidase protein.
 156. Thetransgenic corn plant of claim 128, wherein the non-coding RNA moleculeencoded by the transcribable DNA sequence is a precursor miRNA or siRNAthat is processed or cleaved in the transgenic corn plant to form amature miRNA or siRNA.
 157. The transgenic corn plant of claim 128,wherein the constitutive promoter is selected from the group consistingof: an actin promoter, a CaMV 35S or 19S promoter, a plant ubiquitinpromoter, a plant Gos2 promoter, a FMV promoter, a CMV promoter, a MMVpromoter, a PCLSV promoter, an Emu promoter, a tubulin promoter, anopaline synthase promoter, an octopine synthase promoter, a mannopinesynthase promoter, and a maize alcohol dehydrogenase promoter.
 158. Thetransgenic corn plant of claim 128, wherein the constitutive promotercomprises a DNA sequence that is at least 80% identical to SEQ ID NO:75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ IDNO: 80, SEQ ID NO: 81, SEQ ID NO: 82, or SEQ ID NO:
 83. 159. Thetransgenic corn plant of claim 128, wherein the constitutive promotercomprises a DNA sequence that is at least 90% identical to SEQ ID NO:75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ IDNO: 80, SEQ ID NO: 81, SEQ ID NO: 82, or SEQ ID NO:
 83. 160. Thetransgenic corn plant of claim 128, wherein the constitutive promotercomprises a DNA sequence that is 100% identical to SEQ ID NO: 75, SEQ IDNO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQID NO: 81, SEQ ID NO: 82, or SEQ ID NO:
 83. 161. The transgenic cornplant of claim 128, wherein the recombinant DNA construct is stablyintegrated into the genome of the transgenic plant.
 162. A transgeniccorn plant part or plant cell comprising a recombinant DNA constructcomprising a transcribable DNA sequence encoding a non-coding RNAmolecule, wherein the non-coding RNA molecule comprises a targetingsequence that is: (a) at least 80% complementary to at least 19consecutive nucleotides of a first mRNA molecule encoding a firstendogenous GA oxidase protein in a corn plant, the first endogenous GAoxidase protein being at least 90% identical to SEQ ID NO: 9; and (b) atleast 80% complementary to at least 19 consecutive nucleotides of asecond mRNA molecule encoding a second endogenous GA oxidase protein ina corn plant, the second endogenous GA oxidase protein being at least90% identical to SEQ ID NO: 15; wherein the transcribable DNA sequenceis operably linked to a constitutive promoter.
 163. The transgenic cornplant part or plant cell of claim 162, wherein the non-coding RNAmolecule reduces the expression levels of the first and second mRNAmolecules in at least one tissue of a transgenic corn plant comprisingthe corn plant part or plant cell comprising the recombinant DNAconstruct, relative to a control plant, when the non-coding RNA moleculeis expressed in the transgenic corn plant.
 164. The transgenic cornplant part or plant cell of claim 162, wherein the non-coding RNAmolecule reduces the expression levels of the first and secondendogenous GA oxidase proteins in at least one tissue of a transgeniccorn plant comprising the corn plant part or plant cell comprising therecombinant DNA construct, relative to a control plant, when thenon-coding RNA molecule is expressed in the transgenic corn plant. 165.The transgenic corn plant part or plant cell of claim 162, whereinexpression of the non-coding RNA molecule in the corn plant part orplant cell reduces the level of one or more active GAs in the corn plantpart or plant cell as compared to a control plant part or plant cell.166. The transgenic corn plant part or plant cell of claim 162, whereinthe targeting sequence of the non-coding RNA molecule is at least 90%complementary to at least 19 consecutive nucleotides of the first mRNAmolecule encoding the first endogenous GA20 oxidase protein.
 167. Thetransgenic corn plant part or plant cell of claim 166, wherein thetargeting sequence of the non-coding RNA molecule is 100% complementaryto at least 19 consecutive nucleotides of the first mRNA moleculeencoding the first endogenous GA20 oxidase protein.
 168. The transgeniccorn plant part or plant cell of claim 166, wherein the targetingsequence of the non-coding RNA molecule is at least 90% complementary toat least 19 consecutive nucleotides of SEQ ID NO: 7 or
 8. 169. Thetransgenic corn plant part or plant cell of claim 162, wherein thetargeting sequence of the non-coding RNA molecule is at least 90%complementary to at least 19 consecutive nucleotides of the second mRNAmolecule encoding the second endogenous GA20 oxidase protein.
 170. Thetransgenic corn plant part or plant cell of claim 169, wherein thetargeting sequence of the non-coding RNA molecule is 100% complementaryto at least 19 consecutive nucleotides of the second mRNA moleculeencoding the second endogenous GA20 oxidase protein.
 171. The transgeniccorn plant part or plant cell of claim 169, wherein the targetingsequence of the non-coding RNA molecule is at least 90% complementary toat least 19 consecutive nucleotides of SEQ ID NO: 13 or
 14. 172. Thetransgenic corn plant part or plant cell of claim 162, wherein thetargeting sequence of the non-coding RNA molecule is at least 90%complementary to at least 21 consecutive nucleotides of the first mRNAmolecule encoding the first endogenous GA20 oxidase protein, and is atleast 90% complementary to at least 21 consecutive nucleotides of thesecond mRNA molecule encoding the second GA20 oxidase protein.
 173. Thetransgenic corn plant part or plant cell of claim 162, wherein thetargeting sequence of the non-coding RNA molecule is 100% complementaryto at least 21 consecutive nucleotides of the first mRNA moleculeencoding the first endogenous GA20 oxidase protein and is 100%complementary to at least 21 consecutive nucleotides of the second mRNAmolecule encoding the second GA20 oxidase protein.
 174. The transgeniccorn plant part or plant cell of claim 162, wherein the non-coding RNAmolecule encoded by the transcribable DNA sequence is a precursor miRNAor siRNA that is processed or cleaved in the corn plant part or plantcell to form a mature miRNA or siRNA.
 175. The transgenic corn plantpart or plant cell of claim 162, wherein the constitutive promoter isselected from the group consisting of: an actin promoter, a CaMV 35S or19S promoter, a plant ubiquitin promoter, a plant Gos2 promoter, a FMVpromoter, a CMV promoter, a MMV promoter, a PCLSV promoter, an Emupromoter, a tubulin promoter, a nopaline synthase promoter, an octopinesynthase promoter, a mannopine synthase promoter, and a maize alcoholdehydrogenase promoter.
 176. The transgenic corn plant part or plantcell of claim 162, wherein the constitutive promoter comprises a DNAsequence that is at least 80% identical to SEQ ID NO: 75, SEQ ID NO: 76,SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO:81, SEQ ID NO: 82, or SEQ ID NO:
 83. 177. The transgenic corn plant partor plant cell of claim 162, wherein the constitutive promoter comprisesa DNA sequence that is at least 90% identical to SEQ ID NO: 75, SEQ IDNO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQID NO: 81, SEQ ID NO: 82, or SEQ ID NO:
 83. 178. The transgenic cornplant part or plant cell of claim 162, wherein the constitutive promotercomprises a DNA sequence that is 100% identical to SEQ ID NO: 75, SEQ IDNO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQID NO: 81, SEQ ID NO: 82, or SEQ ID NO:
 83. 179. The transgenic cornplant part or plant cell of claim 162, wherein the recombinant DNAconstruct is stably integrated into the genome of the transgenic cornplant part or plant cell.
 180. A transformation vector comprising therecombinant DNA construct of claim
 111. 181. A DNA molecule comprisingthe recombinant DNA construct of claim
 111. 182. A modified plantproduct made from the transgenic corn plant of claim
 128. 183. Amodified plant product made from the transgenic corn plant part of claim162.
 184. The modified plant product of claim 183, wherein thetransgenic corn plant part is a seed.
 185. A modified plant productcomprising the recombinant DNA construct of claim
 111. 186. Acomposition comprising the recombinant DNA construct of claim
 111. 187.A nonviable corn plant part comprising the recombinant DNA construct ofclaim
 111. 188. A non-regenerable corn plant part comprising therecombinant DNA construct of claim
 111. 189. A nonviable andnon-regenerable corn plant part comprising the recombinant DNA constructof claim
 111. 190. A modified plant product comprising a nonviable ornon-regenerable corn plant part and the recombinant DNA construct ofclaim
 111. 191. A modified corn protoplast comprising the recombinantDNA construct of claim
 111. 192. A modified seed comprising therecombinant DNA construct of claim
 1. 193. The transgenic corn plantpart or plant cell of claim 162, wherein the transgenic corn plant partis a seed.